Neurogenic transcription factors and evolutionarily conserved signalling pathways have been found to be instrumental in the formation of neurons1,2. However, the instructive role of microRNAs (miRNAs) in neurogenesis remains unexplored. We recently discovered that miR-9* and miR-124 instruct compositional changes of SWI/SNF-like BAF chromatin-remodelling complexes, a process important for neuronal differentiation and function3–6. Nearing mitotic exit of neural progenitors, miR-9* and miR-124 repress the BAF53a subunit of the neural-progenitor (np)BAF chromatin-remodelling complex. After mitotic exit, BAF53a is replaced by BAF53b, and BAF45a by BAF45b and BAF45c, which are then incorporated into neuron-specific (n)BAF complexes essential for post-mitotic functions4. Because miR-9/9* and miR-124 also control multiple genes regulating neuronal differentiation and function5,7–13, we proposed that these miRNAs might contribute to neuronal fates. Here we show that expression of miR-9/9* and miR-124 (miR-9/9*-124) in human fibroblasts induces their conversion into neurons, a process facilitated by NEUROD2. Further addition of neurogenic transcription factors ASCL1 and MYT1L enhances the rate of conversion and the maturation of the converted neurons, whereas expression of these transcription factors alone without miR-9/9*-124 was ineffective. These studies indicate that the genetic circuitry involving miR-9/9*-124 can have an instructive role in neural fate determination.

One of the most distinctive steps in the development of the vertebrate nervous system occurs at mitotic exit when cells lose multi-potency and begin to develop stable connections that will persist for a lifetime 1,2 . This transition is accompanied by a switch in ATP-dependent chromatinremodelling mechanisms that appears to coincide with the final mitotic division of neurons. This switch involves the exchange of the BAF53a (also known as ACTL6a) and BAF45a (PHF10) subunits within Swi/Snf-like neural-progenitor-specific BAF (npBAF) complexes for the homologous BAF53b (ACTL6b) and BAF45b (DPF1) subunits within neuron-specific BAF (nBAF) complexes in post-mitotic neurons. The subunits of the npBAF complex are essential for neural-progenitor proliferation, and mice with reduced dosage for the genes encoding its subunits have defects in neural-tube closure similar to those in human spina bifida 3 , one of the most serious congenital birth defects. In contrast, BAF53b and the nBAF complex are essential for an evolutionarily conserved program of post-mitotic neural development and dendritic morphogenesis 4,5 . Here we show that this essential transition is mediated by repression of BAF53a by miR-9* and miR-124. We find that BAF53a repression is mediated by sequences in the 3′ untranslated region corresponding to the recognition sites for miR-9* and miR-124, which are selectively expressed in post-mitotic neurons. Mutation of these sites led to persistent expression of BAF53a and defective activity-dependent dendritic outgrowth in neurons. In addition, overexpression of miR-9* and miR-124 in neural progenitors caused reduced proliferation. Previous studies have indicated that miR-9* and miR-124 are repressed by the repressor-element-1-silencing transcription factor (REST, also known as NRSF) 6 . Indeed, expression of REST in post-mitotic neurons led to derepression of BAF53a, indicating that RESTmediated repression of microRNAs directs the essential switch of chromatin regulatory complexes.The ATP-dependent chromatin-remodelling complexes, typified by the yeast Swi/Snf complex, regulate chromatin assembly and accessibility 7,8 . The mammalian genome encodes nearly 30 different Swi2/Snf2-like ATPases, two of which, BRG1 and BRM are alternative subunits in complexes of 11 subunits termed BAF or mammalian SWI/SNF (mSWI/SNF) 3,4,9-11 . To understand the essential switch in subunit composition of these Correspondence and requests for materials should be addressed to G.R.C. (crabtree@stanford.edu). Supplementary Information is linked to the online version of the paper at www.nature.com/nature. complexes during neural development, we examined 180 kilobases (kb) around the BAF53a gene for transcriptional regulatory regions by replacing the first exon of BAF53a at the start codon with a destabilized nuclear enhanced green fluorescent protein (d2nucEGFP) in a BAF53a-containing bacterial artificial chromosome (BAC). The d2nucEGFP is followed by the stop codon and a 3′ untranslated region (UTR) so that EGFP expression refle...

The Caenorhabditis elegans vulva is an important paradigm for cell-cell interactions in animal development. The fates of six vulval precursor cells are patterned through the action of the epidermal growth factor receptor-mitogen-activated protein kinase (EGFR-MAPK) inductive signaling pathway, which specifies the 1 degrees fate, and the LIN-12/Notch lateral signaling pathway, which specifies the 2 degrees fate. Here, we provide evidence that the inductive signal is spatially graded and initially activates the EGFR-MAPK pathway in the prospective 2 degrees cells. Subsequently, this effect is counteracted by the expression of multiple new negative regulators of the EGFR-MAPK pathway, under direct transcriptional control of the LIN-12-mediated lateral signal.

SUMMARY The promise of using reprogrammed human neurons for disease modeling and regenerative medicine relies on the ability to induce patient-derived neurons with high efficiency and subtype-specificity. We have previously shown that ectopic expression of brain-enriched microRNAs (miRNA), miR-9/9* and miR-124 (miR-9/9*-124), promoted direct conversion of human fibroblasts into neurons. Here we show that co-expression of miR-9/9*-124 with transcription factors enriched in the developing striatum, BCL11B (also known as CTIP2), DLX1, DLX2, MYT1L, can guide the conversion of human postnatal and adult fibroblasts into an enriched population of neurons analogous to striatal medium spiny neurons (MSNs). When transplanted in the mouse brain, the reprogrammed human cells persisted in situ for over 6 months, exhibited membrane properties equivalent to native MSNs and extended projections to the anatomical targets of MSNs. These findings highlight the potential of exploiting the synergism between miR-9/9*-124 and transcription factors to generate specific neuronal subtypes.

We studied a novel function of the presenilins (PS1 and PS2) in governing capacitative calcium entry (CCE), a refilling mechanism for depleted intracellular calcium stores. Abrogation of functional PS1, by either knocking out PS1 or expressing inactive PS1, markedly potentiated CCE, suggesting a role for PS1 in the modulation of CCE. In contrast, familial Alzheimer's disease (FAD)-linked mutant PS1 or PS2 significantly attenuated CCE and store depletion-activated currents. While inhibition of CCE selectively increased the amyloidogenic amyloid beta peptide (Abeta42), increased accumulation of the peptide had no effect on CCE. Thus, reduced CCE is most likely an early cellular event leading to increased Abeta42 generation associated with FAD mutant presenilins. Our data indicate that the CCE pathway is a novel therapeutic target for Alzheimer's disease.

In Huntington’s disease (HD), expansion of CAG codons within the huntingtin gene (HTT) leads to the aberrant formation of protein aggregates and the differential degeneration of striatal medium spiny neurons (MSNs). Modeling HD using patient-specific MSNs has been challenging, as neurons differentiated from induced pluripotent stem cells are free of aggregates and lack an overt cell death phenotype. Here we generated MSNs from HD patient fibroblasts through microRNA-based neuronal conversion, previously shown to bypass the induction of pluripotency and retain age signatures of original fibroblasts. We found that patient MSNs consistently exhibited mutant HTT (mHTT) aggregates, mHTT-dependent DNA damage, mitochondrial dysfunction, and spontaneous degeneration over time in culture. We further provide evidence that erasure of age stored in starting fibroblasts and neuronal conversion of pre-symptomatic HD patient fibroblasts resulted in differential manifestation of cellular phenotypes associated with HD, highlighting the importance of age in modeling late-onset neurological disorders.

Aging is a major risk factor in many forms of late-onset neurodegenerative disorders. The ability to recapitulate age-related characteristics of human neurons in culture will offer unprecedented opportunities to study the biological processes underlying neuronal aging. Here, we show that using a recently demonstrated microRNA-based cellular reprogramming approach, human fibroblasts from postnatal to near centenarian donors can be efficiently converted into neurons that maintain multiple age-associated signatures. Application of an epigenetic biomarker of aging (referred to as epigenetic clock) to DNA methylation data revealed that the epigenetic ages of fibroblasts were highly correlated with corresponding age estimates of reprogrammed neurons. Transcriptome and microRNA profiles reveal genes differentially expressed between young and old neurons. Further analyses of oxidative stress, DNA damage and telomere length exhibit the retention of age-associated cellular properties in converted neurons from corresponding fibroblasts. Our results collectively demonstrate the maintenance of age after neuronal conversion.DOI: http://dx.doi.org/10.7554/eLife.18648.001

SUMMARY Directed reprogramming of human fibroblasts into fully-differentiated neurons requires massive changes in epigenetic and transcriptional states. Induction of a chromatin environment permissive to acquiring neuronal subtype identity is therefore a major barrier to fate conversion. Here we show that the brain-enriched miRNAs miR-9/9* and miR-124 (miR-9/9*-124) trigger reconfiguration of chromatin accessibility, DNA methylation, and mRNA expression to induce a default neuronal state. MiR-9/9*-124-induced neurons (miNs) are functionally excitable and are uncommitted towards specific subtypes yet possess open chromatin at neuronal subtype-specific loci, suggesting such identity can be imparted by additional lineage-specific transcription factors. Consistently, we show ISL1 and LHX3 selectively drive conversion to a highly homogenous population of human spinal cord motor neurons. Taken together, this study shows modular synergism between miRNAs and neuronal subtype-specific transcription factors can drive lineage-specific neuronal reprogramming, thereby providing a general platform for high-efficiency generation of distinct subtypes of human neurons.