Context-dependent expression of a conditionally-inducible form of active Akt

Akt kinases are key signaling components in proliferation-competent and post-mitotic cells. Here, we sought to create a conditionally-inducible form of active Akt for both in vitro and in vivo applications. We fused a ligand-responsive Destabilizing Domain (DD) derived from E. coli dihydrofolate reductase to a constitutively active mutant form of Akt1, Akt(E40K). Prior work indicated that such fusion proteins may be stabilized and induced by a ligand, the antibiotic Trimethoprim (TMP). We observed dose-dependent, reversible induction of both total and phosphorylated/active DD-Akt(E40K) by TMP across several cellular backgrounds in culture, including neurons. Phosphorylation of FoxO4, an Akt substrate, was significantly elevated after DD-Akt(E40K) induction, indicating the induced protein was functionally active. The induced Akt(E40K) protected cells from apoptosis evoked by serum deprivation and was neuroprotective in two cellular models of Parkinson's disease (6-OHDA and MPP+ exposure). There was no significant protection without induction. We also evaluated Akt(E40K) induction by TMP in mouse substantia nigra and striatum after neuronal delivery via an AAV1 adeno-associated viral vector. While there was significant induction in striatum, there was no apparent induction in substantia nigra. To explore the possible basis for this difference, we examined DD-Akt(E40K) induction in cultured ventral midbrain neurons. Both dopaminergic and non-dopaminergic neurons in the cultures showed DD-Akt(E40K) induction after TMP treatment. However, basal DD-Akt(E40K) expression was 3-fold higher for dopaminergic neurons, resulting in a significantly lower induction by TMP in this population. Such findings suggest that dopaminergic neurons may be relatively inefficient in protein degradation, a property that could relate to their lack of apparent DD-Akt(E40K) induction in vivo and to their selective vulnerability in Parkinson's disease. In summary, we generated an inducible, biologically active form of Akt. The degree of inducibility appears to reflect cellular context that will inform the most appropriate applications for this and related reagents

Expression mediated by three partial sequences of the human tyrosine hydroxylase promoter in vivo

The use of viral vectors to transfect postmitotic neurons has provided an important research tool, and it offers promise for treatment of neurologic disease. The utility of vectors is enhanced by the use of selective promoters that permit control of the cellular site of expression. One potential clinical application is in the neurorestorative treatment of Parkinson's disease by the induction of new axon growth. However, many of the genes with an ability to restore axons have oncogenic potential. Therefore, clinical safety would be enhanced by restriction of expression to neurons affected by the disease, particularly dopamine neurons. To achieve this goal we have evaluated in vivo three partial sequences of the promoter for human tyrosine hydroxylase, the rate limiting enzyme in catecholamine synthesis. All sequences induced expression in dopamine neurons. None of them induced expression in glia or in nondopaminergic neurons in striatum or cortex. We conclude that these sequences have potential use for targeting dopamine neurons in research and clinical applications

High-throughput generation of midbrain dopaminergic neuron organoids from reporter human pluripotent stem cells

Summary: Here, we describe a high-throughput 3D differentiation protocol for deriving midbrain dopaminergic neurons from human pluripotent stem cells. The use of organoids has become prevalent in disease modeling, but there is a high demand for more homogeneous cultures. Our approach is advantageous for large-scale production of uniform midbrain organoids that can be maintained in diverse formats, and our reporters allow for sorting of dopaminergic neurons. The maturing long-term organoid cultures can be used as a model for the entire midbrain.For complete details on the use and execution of this protocol, please refer to Ahfeldt et al. (2020)

Generation and characterization of an inducible DD-Akt(E40K).

<p>(A) Design and schematic structure of the DD-Akt(E40K) fusion protein. (B) Scheme depicting mechanism of predicted induction of DD-Akt(E40K) in presence of TMP. (C) Induction of DD-Akt(E40K) by TMP in HEK293 cells as visualized by immunostaining. Cultures were transfected with a bicistronic IRES expression vector (pWPI) expressing the DD-Akt(E40K) fusion protein and eGFP, treated with or without 10 μM TMP for 24 hr and then fixed for immunofluorescence staining. GFP staining indicates cells expressing the vector while HA immunostaining reveals expression of the DD-Akt(E40K) fusion protein. Scale bars indicate 100 μm. (D) Induction of phosphorylated DD-Akt(E40K) by TMP in HEK293 cells as visualized by immunostaining. Cultures and treatment were as in (C) except that immunostaining was for pAkt(pT308) to visualize active Akt. (E) Western immunoblot analysis of total and phosphorylated DD-Akt(E40K) induction by TMP in HEK293 cells. Treatment was as in (C) with the addition that some samples were derived as indicated from cultures transfected with pWPI vector expressing only eGFP. Arrows show positions of endogenous (endo-) and DD-Akt forms. Blots were probed for total Akt and for pAkt(pT308) and pAkt(pS473). ERK 1 and actin served as loading controls. (F) Average fold induction of indicated DD-Akt forms by TMP treatment. HEK293 cultures were prepared as above and quantification of expression determined by western immunoblotting and quantification of relative band densities by ImageJ as in Methods. N = 8–9 experiments each, with duplicate or triplicate wells per condition. Graph shows means with SEM. **p < 0.01, ***p < 0.001, paired t-test +TMP vs.–TMP levels.</p

DD-Akt(E40K) induction is dose-responsive to TMP treatment and reversible.

<p>(A) Western immunoblotting analysis of dose-response of DD-Akt(E40K) induction by TMP. HEK293 cells were transfected with eGFP-only expressing vector (EV) or DD-Akt(E40K) fusion protein expression vector and treated with indicated doses of TMP for 24 hr before analysis by western immunoblotting with the indicated probes. (B) Quantification of DD-Akt(E40K) induction (as determined by western immunoblotting) by various doses of TMP. Values means with SEM. N = 3–4 independent experiments. (C) Time course of DD-Akt(E40K) induction by TMP. HEK293 cells were transfected with DD-Akt(E40K) expression vector, treated with 10 μM TMP for the indicated times and then assessed for total DD-Akt(E40K) levels by western immunoblotting as in (A). Values are means with SEM. N = 2–4 independent experiments. (D) Reversal of DD-Akt(E40K) induction after TMP washout. HEK293 cultures transfected with DD-Akt(E40K) as above were treated with 10 μM TMP for 24 hr, washed free of TMP and harvested for western immunoblotting at the indicated time points. Total DD-Akt(E40K) levels were determined by western immunoblotting as in (A). Data shown are from one experiment.</p

Induction of DD-Akt(E40K) leads to substrate phosphorylation and neuroprotection.

<p>(A) Induction of DD-Akt(E40K) promotes phosphorylation of the putative Akt substrate FoxO4 on pS193. HEK293 cells were transfected with vector expressing eGFP alone (EV) or DD-Akt(E40K) and eGFP, treated with or without 10 μM TMP for 24 hr, and then harvested and analyzed by western immunoblot analysis for the indicated proteins. Total FoxO4 was analyzed on a separate immunoblot and is shown with its own loading controls. Numbers below the bands indicate relative densities (determined by ImageJ), normalized to corresponding ERK1 loading control. (B) Quantification of western immunoblotting data for effect of DD-Akt(E40K) induction on pFoxO4(pS193) levels. Experiments were performed and quantified as in (A). Values are given as mean fold-induction of pFoxO4(pS193) levels under the indicated conditions with SEM. N = 9 independent experiments (7 experiments with no serum present, 2 experiments with serum present). **p<0.01 compared with DD-Akt(E40K)—TMP condition, #p<0.05 compared with empty vector (EV) condition, one-way ANOVA. (C) DD-Akt(E40K) induction protects against serum deprivation. Naïve (NGF-untreated) PC12 cells were infected with lentiviral vectors for overexpression of DD-Akt(E40K) and eGFP or eGFP alone (EV). Four-five days after infection, cultures expressing DD-Akt(E40K) were pre-treated with or without 10 μM TMP for 24 hr and then extensively washed and replated with serum-free medium with or without the continued presence of TMP. EV cultures were deprived of serum in similar fashion and treated either with or without NGF (100 ng/ml). After 16–24 hr of serum deprivation, cells were lysed for quantification of viable nuclei to measure survival. Average infection efficiency was 60%. Data were normalized to survival in NGF-treated cultures and are expressed as means with SEM. *p < 0.05, n.s. vs. EV-NGF; 2-way ANOVA. N = 2–3 independent experiments. (D) Induction of DD-Akt(E40K) protects against the PD mimetic toxin MPP+. Neuronal PC12 cells were infected with lentiviruses for overexpression of DD-Akt(E40K) and eGFP or eGVP alone (EV). Four to five days after infection cultures were pre-treated with or without 10 μM TMP for 24 hr before addition of 0.25 mM MPP+. After 18–24 hr of MPP+ treatment, cells were lysed for quantification of viable nuclei to measure survival. Values are means with SEM and were normalized to cell numbers in cultures infected with EV and not exposed to MPP+. **p < 0.01 vs.–TMP; ##p < 0.01 vs. EV; n.s. vs. EV; 2-way ANOVA. N = 3 independent experiments. (E) Induction of DD-Akt(E40K) protects against the PD mimetic toxin 6-OHDA. Neuronal PC12 cell cultures were established and treated and analyzed as above, except that treatment was with 6-OHDA. Values are means with SEM. ***p < 0.001 vs.–TMP; *p < 0.05 vs.–TMP; ### p < 0.001 vs. Empty Vector; n.s. vs. Empty Vector; 2-way ANOVA. N = 3 independent experiments. (F) TMP does not protect from 6-OHDA in absence of DD-Akt(E40K). Neuronal PC12 cells were pretreated with or without 10 μM TMP for 24 hr before addition of 100 μM or 150 μM 6-OHDA and assessed as above for survival 18–24 hr later. Values are means with SEM. There were no significant effects of TMP on cell survival under any conditions, 2-way ANOVA. n = 2 independent experiments with 3 replicates per condition per experiment.</p

Differential induction of DD-Akt(E40K) within ventral midbrain cultures.

<p>(A) DD-Akt(E40K) induction in ventral midbrain cultures. Ventral midbrain cultures were infected with a lentiviral vector expressing DD-Akt(E40K) and eGFP. 6–10 days post-infection, cultures were treated with or without 10 μM TMP for 4 days and then processed for immunofluorescent staining of tyrosine hydroxylase (TH) to identify dopaminergic neurons, eGFP to identify infected cells, and HA to reveal DD-Akt(E40K). Scale bar indicates 50 μm. (B) HA fluorescence intensity was measured in infected (GFP+) neurons using ImageJ. Dopaminergic vs. non-dopaminergic cells were identified by TH staining. Values are mean fluoresence intensity in arbitary units (A.U.) with SEM. n = 7–8 TH+ neurons measured for each condition, 12–14 non-TH neurons measured for each condition. *p < 0.05: TH, -TMP vs. + TMP; ****p < 0.0001: non-TH, -TMP vs. + TMP; #p < 0.05: TH, -TMP vs. non-TH, -TMP, 2-way ANOVA.</p

Regional inducibility of DD-Akt(E40K) in the mouse brain.

<p>(A) Scheme of experimental design. Adult C57BL/6 mice were subjected to unilateral AAV-DD-Akt(E40K)-Flag injections into the SNpc or striatum. At 3 weeks post-viral injection, the mice were treated with or without 1 mg/ml TMP in their drinking water for 3 weeks and then their brains were harvested and processed for Flag immunostaining. (B) Representative images of Flag immunostained neurons in the SNpc of both TMP-untreated and TMP-treated mice. (C) Stereological quantification of total numbers of Flag-stained neurons in the SNpc of mice treated as above with or without TMP. The data represent total Flag-positive neuron numbers in the SNpc per mouse. Mean values are shown with SEM. n = 7 mice for -TMP, n = 6 mice for +TMP. n.s., determined by unpaired t-test. (D) Representative images of Flag immunostaining in the striatum of both TMP-untreated and TMP-treated mice. Staining in the medial striatum is outlined by a black rectangle and shown at higher magnification in the inset; the external capsule (EC) and needle track (NT) staining is indicated by brackets. (E) Stereological quantification of total numbers of Flag-stained neurons in the striata of mice treated as above with or without TMP. The data represent total Flag-positive neuron numbers in the entire striatum per mouse. Mean values are shown with SEM. n = 5 mice for -TMP, n = 8 mice for +TMP. *p = 0.03, unpaired t-test (+TMP vs -TMP).</p

Novel human pluripotent stem cell-derived hypothalamus organoids demonstrate cellular diversity

Summary: The hypothalamus is a region of the brain that plays an important role in regulating body functions and behaviors. There is a growing interest in human pluripotent stem cells (hPSCs) for modeling diseases that affect the hypothalamus. Here, we established an hPSC-derived hypothalamus organoid differentiation protocol to model the cellular diversity of this brain region. Using an hPSC line with a tyrosine hydroxylase (TH)-TdTomato reporter for dopaminergic neurons (DNs) and other TH-expressing cells, we interrogated DN-specific pathways and functions in electrophysiologically active hypothalamus organoids. Single-cell RNA sequencing (scRNA-seq) revealed diverse neuronal and non-neuronal cell types in mature hypothalamus organoids. We identified several molecularly distinct hypothalamic DN subtypes that demonstrated different developmental maturities. Our in vitro 3D hypothalamus differentiation protocol can be used to study the development of this critical brain structure and can be applied to disease modeling to generate novel therapeutic approaches for disorders centered around the hypothalamus