Direct conversion of mouse astrocytes into neural progenitor cells and specific lineages of neurons

Background:Cell replacement therapy has been envisioned as a promising treatment for neurodegenerative diseases.Due to the ethical concerns of ESCs-derived neural progenitor cells(NPCs)and tumorigenic potential of iPSCs,reprogramming of somatic cells directly into multipotent NPCs has emerged as a preferred approach for cell transplantation.Methods:Mouse astrocytes were reprogrammed into NPCs by the overexpression of transcription factors(TFs)Foxg1,Sox2,and Brn2.The generation of subtypes of neurons was directed by the force expression of cell-type specific TFs Lhx8 or Foxa2/Lmx1a.Results:Astrocyte-derived induced NPCs(AiNPCs)share high similarities,including the expression of NPC-specific genes,DNA methylation patterns,the ability to proliferate and differentiate,with the wild type NPCs.The AiNPCs are committed to the forebrain identity and predominantly differentiated into glutamatergic and GABAergic neuronal subtypes.Interestingly,additional overexpression of TFs Lhx8 and Foxa2/Lmx1a in AiNPCs promoted cholinergic and dopaminergic neuronal differentiation,respectively.Conclusions:Our studies suggest that astrocytes can be converted into AiNPCs and lineage-committed AiNPCs can acquire differentiation potential of other lineages through forced expression of specific TFs.Understanding the impact of the TF sets on the reprogramming and differentiation into specific lineages of neurons will provide valuable strategies for astrocyte-based cell therapy in neurodegenerative diseases.

Serial deletion reveals structural basis and stability for the core enzyme activity of human glutaminase 1 isoforms:relevance to excitotoxic neurodegeneration

Background:Glutaminase 1 is a phosphate-activated metabolic enzyme that catalyzes the first step of glutaminolysis,which converts glutamine into glutamate.Glutamate is the major neurotransmitter of excitatory synapses,executing important physiological functions in the central nervous system.There are two isoforms of glutaminase 1,KGA and GAC,both of which are generated through alternative splicing from the same gene.KGA and GAC both transcribe 1–14 exons in the N-terminal,but each has its unique C-terminal in the coding sequence.We have previously identified that KGA and GAC are differentially regulated during inflammatory stimulation and HIV infection.Furthermore,glutaminase 1 has been linked to brain diseases such as amyotrophic lateral sclerosis,Alzheimer’s disease,and hepatic encephalopathy.Core enzyme structure of KGA and GAC has been published recently.However,how other coding sequences affect their functional enzyme activity remains unclear.Methods:We cloned and performed serial deletions of human full-length KGA and GAC from the N-terminal and the C-terminal at an interval of approximately 100 amino acids(AAs).Prokaryotic expressions of the mutant glutaminase 1 protein and a glutaminase enzyme activity assay were used to determine if KGA and GAC have similar efficiency and efficacy to convert glutamine into glutamate.Results:When 110 AAs or 218 AAs were deleted from the N-terminal or when the unique portions of KGA and GAC that are beyond the 550 AA were deleted from the C-terminal,KGA and GAC retained enzyme activity comparable to the full length proteins.In contrast,deletion of 310 AAs or more from N-terminal or deletion of 450 AAs or more from C-terminal resulted in complete loss of enzyme activity for KGA/GAC.Consistently,when both Nand C-terminal of the KGA and GAC were removed,creating a truncated protein that expressed the central 219 AA-550 AA,the protein retained enzyme activity.Furthermore,expression of the core 219 AA-550 AA coding sequence in cells increased extracellular glutamate concentrations to levels comparable to those of full-length KGA and GAC expressions,suggesting that the core enzyme activity of the protein lies within the central 219 AA-550 AA.Full-length KGA and GAC retained enzyme activities when kept at 4°C.In contrast,219 AA-550 AA truncated protein lost glutaminase activities more readily compared with full-length KGA and GAC,suggesting that the Nterminal and C-terminal coding regions are required for the stability KGA and GAC.Conclusions:Glutaminase isoforms KGA and GAC have similar efficacy to catalyze the conversion of glutamine to glutamate.The core enzyme activity of glutaminase 1 protein is within the central 219 AA-550 AA.The N-terminal and C-terminal coding regions of KGA and GAC help maintain the long-term activities of the enzymes.