Cellular differentiation and lineage commitment are considered robust and irreversible processes during development. Recent work has shown that mouse and human fibroblasts can be reprogrammed to a pluripotent state with a combination of four transcription factors. This raised the question of whether transcription factors could directly induce other defined somatic cell fates, and not only an undifferentiated state. We hypothesized that combinatorial expression of neural lineage-specific transcription factors could directly convert fibroblasts into neurons. Starting from a pool of nineteen candidate genes, we identified a combination of only three factors, Ascl1, Brn2, and Myt1l, that suffice to rapidly and efficiently convert mouse embryonic and postnatal fibroblasts into functional neurons in vitro. These induced neuronal (iN) cells express multiple neuron-specific proteins, generate action potentials, and form functional synapses. Generation of iN cells from non-neural lineages could have important implications for studies of neural development, neurological disease modeling, and regenerative medicine.

Summary Somatic cell nuclear transfer, cell fusion, or expression of lineage-specific factors have been shown to induce cell-fate changes in diverse somatic cell types1–12. We recently observed that forced expression of a combination of three transcription factors, Brn2 (also known as Pou3f2), Ascl1, and Myt1l can efficiently convert mouse fibroblasts into functional induced neuronal (iN) cells13. Here, we show that the same three factors can generate functional neurons from human pluripotent stem cells as early as 6 days after transgene activation. When combined with the basic helix-loop-helix transcription factor NeuroD1, these factors could also convert fetal and postnatal human fibroblasts into iN cells displaying typical neuronal morphologies and expressing multiple neuronal markers, even after downregulation of the exogenous transcription factors. Importantly, the vast majority of human iN cells were able to generate action potentials and many matured to receive synaptic contacts when co-cultured with primary mouse cortical neurons. Our data demonstrate that non-neural human somatic cells, as well as pluripotent stem cells, can be directly converted into neurons by lineage-determining transcription factors. These methods may facilitate robust generation of patient-specific human neurons for in vitro disease modeling or future applications in regenerative medicine.

Ca2+-triggered synchronous neurotransmitter release is well described, but asynchronous release-in fact, its very existence-remains enigmatic. Here we report a quantitative description of asynchronous neurotransmitter release in calyx-of-Held synapses. We show that deletion of synaptotagmin 2 (Syt2) in mice selectively abolishes synchronous release, allowing us to study pure asynchronous release in isolation. Using photolysis experiments of caged Ca2+, we demonstrate that asynchronous release displays a Ca2+ cooperativity of approximately 2 with a Ca2+ affinity of approximately 44 microM, in contrast to synchronous release, which exhibits a Ca2+ cooperativity of approximately 5 with a Ca2+ affinity of approximately 38 muM. Our results reveal that release triggered in wild-type synapses at low Ca2+ concentrations is physiologically asynchronous, and that asynchronous release completely empties the readily releasable pool of vesicles during sustained elevations of Ca2+. We propose a dual-Ca2+-sensor model of release that quantitatively describes the contributions of synchronous and asynchronous release under conditions of different presynaptic Ca2+ dynamics.

Trans-SNARE complexes catalyze fast synaptic vesicle fusion and bind complexin, but the function of complexin binding to SNARE complexes remains unclear. Here we show that in neuronal synapses, complexin simultaneously suppressed spontaneous fusion and activated fast Ca 2+ -evoked fusion. The dual function of complexin required SNARE binding, and additionally involved distinct N-terminal sequences of complexin that localize to the point where trans-SNARE complexes insert into the fusing membranes, suggesting that complexin controls the force that trans-SNARE complexes apply onto the fusing membranes. Consistent with this hypothesis, a mutation in the membrane insertion sequence of the v-SNARE synaptobrevin/VAMP phenocopied the complexin loss-of-function state without impairing complexin-binding to SNARE complexes. Thus, complexin probably activates and clamps the force-transfer from assembled trans-SNARE complexes onto fusing membranes.Synaptic vesicle fusion is driven by assembly of trans-SNARE complexes (or SNAREpins) from syntaxin-1 and SNAP-25 on the plasma membrane, and synaptobrevin/ VAMP on the vesicle membrane [1][2][3]. Ca 2+ then triggers fast synchronous synaptic vesicle fusion by binding to the Ca 2+ -sensor synaptotagmin [4][5][6]. Besides SNARE proteins and synaptotagmin, fast Ca 2+ -triggered fusion requires complexin [7]. Complexin is composed of short N-and C-terminal sequences and two central α-helices. Complexin binds to SNARE complexes via its central α-helix, which inserts in an anti-parallel orientation into a groove formed by synaptobrevin/VAMP and syntaxin-1 [8,9]. Although multiple approaches have revealed an essential role of complexin in synaptic fusion [7,[10][11][12][13][14][15], the nature of this role remains unclear. In vertebrate autapses, deletion of complexin selectively impairs fast synchronous neurotransmitter release without changing asynchronous or spontaneous release [7,10]. In in vitro fusion assays, conversely, addition of complexin causes a general block of SNARE-dependent fusion, indicating that complexin is a SNARE clamp [11][12][13][14]. In Drosophila neuromuscular synapses, deletion of complexin produces a >20-fold increase in spontaneous release but only a small decrease in evoked release [15]. Thus, the role of complexin in fusion is unclear. Moreover, even the importance of complexin SNARE- [16,17]. Here we addressed these questions with two complementary approaches -RNAi-dependent knockdown of complexin with rescue, and replacing wild-type synaptobrevin with specific mutants using synaptobrevin knockout (KO) mice [18].We knocked down complexin expression in cultured cortical neurons using an shRNA that targets both complexin-1 and -2, the only complexin isoforms significantly expressed in these neurons [19]. For this purpose, we used lentiviruses that simultaneously synthesize the complexin shRNA and either GFP, wild-type complexin-1, or 4M-mutant complexin-1 that is unable to bind to SNARE complexes (19,20). Lentivirus expressing GFP without the shRNA ...