Novel Green Fluorescent Polyamines to Analyze ATP13A2 and ATP13A3 Activity in the Mammalian Polyamine Transport System.

Cells acquire polyamines putrescine (PUT), spermidine (SPD) and spermine (SPM) via the complementary actions of polyamine uptake and synthesis pathways. The endosomal P5B-type ATPases ATP13A2 and ATP13A3 emerge as major determinants of mammalian polyamine uptake. Our biochemical evidence shows that fluorescently labeled polyamines are genuine substrates of ATP13A2. They can be used to measure polyamine uptake in ATP13A2- and ATP13A3-dependent cell models resembling radiolabeled polyamine uptake. We further report that ATP13A3 enables faster and stronger cellular polyamine uptake than does ATP13A2. We also compared the uptake of new green fluorescent PUT, SPD and SPM analogs using different coupling strategies (amide, triazole or isothiocyanate) and fluorophores (symmetrical BODIPY, BODIPY-FL and FITC). ATP13A2 promotes the uptake of various SPD and SPM analogs, whereas ATP13A3 mainly stimulates the uptake of PUT and SPD conjugates. However, the polyamine linker and coupling position on the fluorophore impacts the transport capacity, whereas replacing the fluorophore affects polyamine selectivity. The highest uptake in ATP13A2 or ATP13A3 cells is observed with BODIPY-FL-amide conjugated to SPD, whereas BODIPY-PUT analogs are specifically taken up via ATP13A3. We found that P5B-type ATPase isoforms transport fluorescently labeled polyamine analogs with a distinct structure-activity relationship (SAR), suggesting that isoform-specific polyamine probes can be designed.

Evaluation of WGA-Cre-dependent topological transgene expression in the rodent brain.

Novel neuromodulation techniques in the field of brain research, such as optogenetics, prompt to target specific cell populations. However, not every subpopulation can be distinguished based on brain area or activity of specific promoters, but rather on topology and connectivity. A fascinating tool to detect neuronal circuitry is based on the transsynaptic tracer, wheat germ agglutinin (WGA). When expressed in neurons, it is transported throughout the neuron, secreted, and taken up by synaptically connected neurons. Expression of a WGA and Cre recombinase fusion protein using a viral vector technology in Cre-dependent transgenic animals allows to trace neuronal network connections and to induce topological transgene expression. In this study, we applied and evaluated this technology in specific areas throughout the whole rodent brain, including the hippocampus, striatum, substantia nigra, and the motor cortex. Adeno-associated viral vectors (rAAV) encoding the WGA-Cre fusion protein under control of a CMV promoter were stereotactically injected in Rosa26-STOP-EYFP transgenic mice. After 6 weeks, both the number of transneuronally labeled YFP+/mCherry- cells and the transduced YFP+/mCherry+ cells were quantified in the connected regions. We were able to trace several connections using WGA-Cre transneuronal labeling; however, the labeling efficacy was region-dependent. The observed transneuronal labeling mostly occurred in the anterograde direction without the occurrence of multi-synaptic labeling. Furthermore, we were able to visualize a specific subset of newborn neurons derived from the subventricular zone based on their connectivity.

rAAV-CFTRΔR Rescues the Cystic Fibrosis Phenotype in Human Intestinal Organoids and Cystic Fibrosis Mice.

RATIONALE: Gene therapy holds promise for a curative mutation-independent treatment applicable to all patients with cystic fibrosis (CF). The various viral vector-based clinical trials conducted in the past have demonstrated safety and tolerance of different vectors, but none have led to a clear and persistent clinical benefit. Recent clinical breakthroughs in recombinant adeno-associated viral vector (rAAV)-based gene therapy encouraged us to reexplore an rAAV approach for CF. OBJECTIVES: We evaluated the preclinical potential of rAAV gene therapy for CF to restore chloride and fluid secretion in two complementary models: intestinal organoids derived from subjects with CF and a CF mouse model, an important milestone toward the development of a clinical rAAV candidate for CF gene therapy. METHODS: We engineered an rAAV vector containing a truncated CF transmembrane conductance regulator (CFTRΔR) combined with a short promoter (CMV173) to ensure optimal gene expression. A rescue in chloride and fluid secretion after rAAV-CFTRΔR treatment was assessed by forskolin-induced swelling in CF transmembrane conductance regulator (CFTR)-deficient organoids and by nasal potential differences in ΔF508 mice. MEASUREMENTS AND MAIN RESULTS: rAAV-CFTRΔR transduction of human CFTR-deficient organoids resulted in forskolin-induced swelling, indicating a restoration of CFTR function. Nasal potential differences demonstrated a clear response to low chloride and forskolin perfusion in most rAAV-CFTRΔR-treated CF mice. CONCLUSIONS: Our study provides robust evidence that rAAV-mediated gene transfer of a truncated CFTR functionally rescues the CF phenotype across the nasal mucosa of CF mice and in patient-derived organoids. These results underscore the clinical potential of rAAV-CFTRΔR in offering a cure for all patients with CF in the future.