In Parkinson's disease, progressive dysfunction and degeneration of dopamine neurons in the ventral midbrain cause life-changing symptoms. Neuronal degeneration has diverse causes in Parkinson's, including non-cell autonomous mechanisms mediated by astrocytes. Throughout the CNS, astrocytes are essential for neuronal survival and function, as they maintain metabolic homeostasis in the neural environment. Astrocytes interact with the immune cells of the CNS, microglia, to modulate neuroinflammation, which is observed from the earliest stages of Parkinson's, and has a direct impact on the progression of its pathology. In diseases with a chronic neuroinflammatory element, including Parkinson's, astrocytes acquire a neurotoxic phenotype, and thus enhance neurodegeneration. Consequently, astrocytes are a potential therapeutic target to slow or halt disease, but this will require a deeper understanding of their properties and roles in Parkinson's. Accurate models of human ventral midbrain astrocytes for in vitro study are therefore urgently required. We have developed a protocol to generate high purity cultures of ventral midbrain-specific astrocytes (vmAstros) from hiPSCs that can be used for Parkinson's research. vmAstros can be routinely produced from multiple hiPSC lines, and express specific astrocytic and ventral midbrain markers. This protocol is scalable, and thus suitable for high-throughput applications, including for drug screening. Crucially, the hiPSC derived-vmAstros demonstrate immunomodulatory characteristics typical of their in vivo counterparts, enabling mechanistic studies of neuroinflammatory signaling in Parkinson's.

h4>SUMMARY/h4> Parkinson disease is characterized by loss of dopamine neurons in the substantia nigra. As with other neurodegenerative diseases, no disease-modifying treatments exist. While most treatment objectives aim to prevent neuronal loss or protect vulnerable neuronal circuits, an important alternative is to replace lost neurons to reconstruct disrupted circuits. Herein we report an efficient single-step conversion of isolated mouse and human astrocytes into functional neurons by depleting the RNA binding protein PTB. Applying this approach to mice with a chemically induced Parkinson’s phenotype, we provide evidence that disease manifestations can be potently reversed through converting astrocytes into new substantia nigral neurons, effectively restoring dopamine levels via reestablishing the nigrostriatal dopamine pathway. We further demonstrate similar disease reversal with a therapeutically feasible approach using antisense oligonucleotides to transiently suppress PTB. These findings identify a generalizable therapeutic strategy for treating neurodegenerative disorders through replacing lost neurons in the brain.