A general approach for stabilizing nanobodies for intracellular expression.

Conventional antibodies and their derived fragments are difficult to deploy against intracellular targets in live cells, due to their bulk and structural complexity. Nanobodies provide an alternative modality, with well-documented examples of intracellular expression. Despite their promise as intracellular reagents, there has not been a systematic study of nanobody intracellular expression.

Retinoic acid signaling mediates peripheral cone photoreceptor survival in a mouse model of retina degeneration.

Retinitis Pigmentosa (RP) is a progressive, debilitating visual disorder caused by mutations in a diverse set of genes. In both humans with RP and mouse models of RP, rod photoreceptor dysfunction leads to loss of night vision, and is followed by secondary cone photoreceptor dysfunction and degeneration, leading to loss of daylight color vision. A strategy to prevent secondary cone death could provide a general RP therapy to preserve daylight color vision regardless of the underlying mutation.

Probe-Seq: Method for RNA Sequencing of Specific Cell Types from Animal Tissue.

Most organs and tissues are composed of many types of cells. To characterize cellular state, various transcription profiling approaches are currently available, including whole-tissue bulk RNA sequencing, single cell RNA sequencing (scRNA-Seq), and cell type-specific RNA sequencing. What is missing in this repertoire is a simple, versatile method for bulk transcriptional profiling of cell types for which cell type-specific genetic markers or antibodies are not readily available.

Erratum: Correction Notice: Probe-Seq: Method for RNA Sequencing of Specific Cell Types from Animal Tissue.

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Probe-Seq enables transcriptional profiling of specific cell types from heterogeneous tissue by RNA-based isolation.

Recent transcriptional profiling technologies are uncovering previously-undefined cell populations and molecular markers at an unprecedented pace. While single cell RNA (scRNA) sequencing is an attractive approach for unbiased transcriptional profiling of all cell types, a complementary method to isolate and sequence specific cell populations from heterogeneous tissue remains challenging. Here, we developed Probe-Seq, which allows deep transcriptional profiling of specific cell types isolated using RNA as the defining feature.

FIN-Seq: transcriptional profiling of specific cell types from frozen archived tissue of the human central nervous system.

Thousands of frozen, archived tissue samples from the human central nervous system (CNS) are currently available in brain banks. As recent developments in RNA sequencing technologies are beginning to elucidate the cellular diversity present within the human CNS, it is becoming clear that an understanding of this diversity would greatly benefit from deeper transcriptional analyses. Single cell and single nucleus RNA profiling provide one avenue to decipher this heterogeneity.

Adult axolotls can regenerate original neuronal diversity in response to brain injury.

The axolotl can regenerate multiple organs, including the brain. It remains, however, unclear whether neuronal diversity, intricate tissue architecture, and axonal connectivity can be regenerated; yet, this is critical for recovery of function and a central aim of cell replacement strategies in the mammalian central nervous system. Here, we demonstrate that, upon mechanical injury to the adult pallium, axolotls can regenerate several of the populations of neurons present before injury.

Highly multiplexed subcellular RNA sequencing in situ.

Understanding the spatial organization of gene expression with single-nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here, we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked complementary DNA (cDNA) amplicons are sequenced within a biological sample. Using 30-base reads from 8102 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay.

Development-inspired reprogramming of the mammalian central nervous system.

In 2012, John Gurdon and Shinya Yamanaka shared the Nobel Prize for the demonstration that the identity of differentiated cells is not irreversibly determined but can be changed back to a pluripotent state under appropriate instructive signals. The principle that differentiated cells can revert to an embryonic state and even be converted directly from one cell type into another not only turns fundamental principles of development on their heads but also has profound implications for regenerative medicine. Replacement of diseased tissue with newly reprogrammed cells and modeling of human disease are concrete opportunities.

FOXO3 determines the accumulation of α-synuclein and controls the fate of dopaminergic neurons in the substantia nigra.

Parkinson's disease (PD) is characterized by the selective degeneration of neuronal populations presumably due to pathogenic interactions between aging and predisposing factors such as increased levels of α-synuclein. Here, we genetically modulate the activity of the transcription factor Forkhead box protein O3 (FOXO3) in adult nigral dopaminergic neurons using viral vectors and explore how this determinant of longevity impacts on neuronal fate in normal and diseased conditions. We find that dopaminergic neurons are particularly vulnerable to changes in FOXO3 activity in the substantia nigra.

Reshaping the brain: direct lineage conversion in the nervous system.

During embryonic development, cells in an uncommitted pluripotent state undergo progressive epigenetic changes that lock them into a final restrictive differentiated state. However, recent advances have shown that not only is it possible for a fully differentiated cell to revert back to a pluripotent state, a process called nuclear reprogramming, but also that differentiated cells can be directly converted from one class into another without generating progenitor intermediates, a process known as direct lineage conversion. In this review, we discuss recent progress made in direct lineage reprogramming of differentiated cells into neurons and discuss some of the therapeutic implications of the findings.

Excessive alcohol consumption is blocked by glial cell line-derived neurotrophic factor.

We previously found that activation of the glial cell line-derived neurotrophic factor (GDNF) pathway in the ventral tegmental area (VTA) reduces moderate alcohol (ethanol) intake in a rat operant self-administration paradigm. Here, we set out to assess the effect of GDNF in the VTA on excessive voluntary consumption of ethanol. Long-Evans rats were trained to drink large quantities of a 20% ethanol solution in an intermittent-access two-bottle choice drinking paradigm.