Stepwise Protocols for Converting Astrocytes to Neurons in vitro and in Mouse Brain by Depleting Polypyrimidine Tract Binding Protein PTB

We recently develop an ecient single-step strategy to convert isolated mouse and human astrocytes into functional neurons by depleting the RNA binding protein PTB in isolated astrocytes in culture and directly in mouse brain. We show progressive conversion of astrocytes into new neurons that can innervate into endogenous neural circuits. Focusing on midbrain, we demonstrate ecient conversion of nigral astrocytes into dopaminergic neurons whose axons reconstruct the nigro-striatal circuit. Signicantly, re-innervation of striatum is accompanied by restoration of dopamine levels and rescue of motor decits. Herein, we present key methods employed in this study as stepwise protocols.


Introduction
Regenerative medicine promises to address disorders that feature cell loss 1 . Exploring the cellular plasticity of differentiated somatic cells 2 , switching cell fate in situ has become an important alternative approach 3 . In the central nervous system, glial cells exhibit remarkable plasticity for reprogramming 4 , which has been leveraged to generate new neurons that lead to behavioral bene ts in various disease models 5,6 .
In comparison with most in vivo reprogramming approaches that rely on one or more lineagespeci c transcription factors (TFs), we recently elucidate roles for the RNA binding protein PTB and its neuronal analog nPTB in neuronal induction and maturation and show that sequential depletion of these RNA binding proteins are able to e ciently convert both mouse and human broblasts to functional neurons 7,8 . We have now used this strategy to convert astrocytes to functional neurons in vitro and directly in mouse brain by taking advantage of inactive PTB-regulated loop but active nPTB-regulated loop in astrocytes, which enables their conversion to functional neurons by depleting PTB alone.
By injecting AAV-shPTB into mouse midbrain, we show that a signi cant fraction of transduced astrocytes are converted into dopaminergic (DA) neurons in substantia nigra, with axons targeted to striatum. Extensive immunochemical evidence and electrophysiological recording demonstrate the function of re-innervated neurons. Applying this strategy to a chemically induced mouse Parkinson's disease (PD) model, we demonstrate that PTB depletion-induced DA neurons potently reverse PD-relevant motor phenotypes. Our ndings thus illuminate a new strategy to treat PD and perhaps other neurodegenerative diseases. Here, we provide stepwise protocols for key methods employed in this study.

7)
To express RFP and shPTB under the GFAP promoter, take a segment containing oxed/off RFP and shPTB to replace EGFP in the AAV-GFAP-EGFP vector (Addgene, #50473) between the Sal I and Hind III sites. 80) Package AAV2 viral particles in co-transfected HEK293T cells with the other two plasmids: pAAV-RC and pAAV-Helper (Agilent Genomics) 10 . 9) After harvest, purify viral particles with a heparin column (GE HEALTHCARE BIOSCIENCES) and then concentrate with an Ultra-4 centrifugal lter unit (Amicon, 100,000 molecular weight cutoff).

Trans-differentiation of isolated astrocytes to neurons in vitro
1) Isolate mouse astrocytes from postnatal (P4~P5) pups: Dissect cortical tissue from cortex or midbrain and incubate with Trypsin before plating onto dishes coated with Poly-D-lysine (Sigma). Purchase human astrocytes from Cell Applications, which were taken from cerebral cortex at the gestational age of 19 weeks.
3. Characterization of converted neurons by immunostaining 1). Grow cells on glass slides and xed with 4% paraformaldehyde (PFA, Affymetrix) for 15 min at room temperature followed by permeabilization with 0.1% Triton X-100 in PBS for 15 min on ice.
2). After washing twice with PBS, block cells in PBS containing 3% BSA for 1 hr at room temperature.
3). Incubate xed cells with primary antibodies overnight at 4°C in PBS containing 3% BSA. 4). After washing twice with PBS, include cells with secondary antibodies conjugated to Alexa Fluor 488, Alexa 546, Alexa 594 or Alexa 647 (1:500, Molecular Probes) for 1 hr. 5). Add 300 nM DAPI in PBS and wait for 20 min at room temperature to label nuclei. 6). After washing three times with PBS, apply the Fluoromount-G mounting medium onto the glass slides, and take images under Olympus FluoView FV1000. 7). For staining brain sections, sacri ce mice with CO2 and immediately perfuse them, rst with 15~20mL saline (0.9% NaCl) and then with 15 mL 4% PFA in PBS to x tissue. 8). Fix whole brains in 4% PFA overnight at 4°C, and then cut into 14~18mm sections on a cryostat (Leica). 9). Before staining, incubate brain sections with sodium citrate buffer (10 mM Sodium citrate, 0.05% Tween 20, pH 6.0) for 15 min at 95°C for antigen retrieval. 10). Treat brain slides with 5% normal donkey serum and 0.3% Triton X-100 in PBS for 1 hr at room temperature. Perform the rest of steps as with cultured cells on coverslips.
4. Quanti cation of neuronal cell body and ber density 1). Sample coronal sections across midbrain at intervals of 120~140 μm for immunostaining of TH and RFP.
2). Calculate the total number (Nt) of cell types by the stereological method correcting with the Abercrombie formula, as described 12 , using the formula: Nt = Ns*(St/Ss)*M/(M+D), where Ns is the number of neurons counted, St is the total number of sections in the brain region, Ss is the number of sections sampled, M is the thickness of section, and D is the average diameter of counted cells, as previously described 13,14 .
3). Quantify RFP+ and RFP+TH+ bers using a previously published sphere method 15  2). Acquire data with pClamp 10.0 or Igor 4.04 software and analyze data with MatLab v2009b.
3). For recording on neurons converted from mouse astrocytes in vitro, remove small molecules from medium 1 week before patch clamp recording. 6. Ipsilateral lesion with 6-OHDA and stereotaxic injection 1). Use adult wt and GFAP-Cre mice at age of postnatal day 30 to perform surgery to induce lesion.
2). For rotation test, record apomorphine-induced rotations in mice after intraperitoneal injection of apomorphine (Sigma, 0.5 mg/kg) under a live video system.
3). Inject mice with apomorphine (0.5 mg/kg) on two separate days prior to performing the rotation test (for example, if the test was to be performed on Friday, the mouse would be rst injected on Monday and Wednesday), which aimed to prevent a 'wind-up' effect that could obscure the nal results. 4). Measure rotation 5 min following the injection for 10 min, as previously described 19,20 and only count full-body turns. 5). Perform rotation test induced by D-amphetamine (Sigma, 5 mg/kg) in the same system, as previously described 21,22 . 6). Express the data as net contralateral or ipsilateral turns per min.
Page 11/13 7). To perform the cylinder test, place mouse into a glass cylinder (diameter 19 cm, height 25 cm), with mirrors placed behind for a full view of all touches, as previously described 19,23 . 8). Record mice under a live video system and no habituation of the mice to the cylinder performed before recording. 9). Use a frame-by-frame video player (KMPlayer version 4.0.7.1) for scoring. Only count wall touches independently with the ipsilateral or the contralateral forelimb. Do not include simultaneous wall touches (touched executed with both paws at the same time) in the analysis. 10). Express data are as a percentage of ipsilateral touches in total touches. 11). For chemogenetic experiments, perform cylinder tests 21-28 days after 6-OHDA induced lesion and 2 months after the delivery of AAV-hM4Di-shPTB.