Augmenting fibronectin levels in injured adult CNS promotes axon regeneration in vivo.

In an attempt to repair injured central nervous system (CNS) nerves/tracts, immune cells are recruited into the injury site, but endogenous response in adult mammals is insufficient for promoting regeneration of severed axons. Here, we found that a portion of retinal ganglion cell (RGC) CNS projection neurons that survive after optic nerve crush (ONC) injury are enriched for and upregulate fibronectin (Fn)-interacting integrins Itga5 and ItgaV, and that Fn promotes long-term survival and long-distance axon regeneration of a portion of axotomized adult RGCs in culture. We then show that, Fn is developmentally downregulated in the axonal tracts of optic nerve and spinal cord, but injury-activated macrophages/microglia upregulate Fn while axon regeneration-promoting zymosan augments their recruitment (and thereby increases Fn levels) in the injured optic nerve. Finally, we found that Fn's RGD motif, established to interact with Itga5 and ItgaV, promotes long-term survival and long-distance axon regeneration of adult RGCs after ONC in vivo, with some axons reaching the optic chiasm when co-treated with Rpl7a gene therapy. Thus, experimentally augmenting Fn levels in the injured CNS is a promising approach for therapeutic neuroprotection and axon regeneration of at least a portion of neurons.

Pten inhibition dedifferentiates long-distance axon-regenerating intrinsically photosensitive retinal ganglion cells and upregulates mitochondria-associated Dynlt1a and Lars2.

Central nervous system projection neurons fail to spontaneously regenerate injured axons. Targeting developmentally regulated genes in order to reactivate embryonic intrinsic axon growth capacity or targeting pro-growth tumor suppressor genes such as Pten promotes long-distance axon regeneration in only a small subset of injured retinal ganglion cells (RGCs), despite many RGCs regenerating short-distance axons. A recent study identified αRGCs as the primary type that regenerates short-distance axons in response to Pten inhibition, but the rare types which regenerate long-distance axons, and cellular features that enable such response, remained unknown. Here, we used a new method for capturing specifically the rare long-distance axon-regenerating RGCs, and also compared their transcriptomes with embryonic RGCs, in order to answer these questions. We found the existence of adult non-α intrinsically photosensitive M1 RGC subtypes that retained features of embryonic cell state, and showed that these subtypes partially dedifferentiated towards an embryonic state and regenerated long-distance axons in response to Pten inhibition. We also identified Pten inhibition-upregulated mitochondria-associated genes, Dynlt1a and Lars2, which promote axon regeneration on their own, and thus present novel therapeutic targets.

Post-injury born oligodendrocytes incorporate into the glial scar and contribute to the inhibition of axon regeneration.

Failure of central nervous system projection neurons to spontaneously regenerate long-distance axons underlies irreversibility of white matter pathologies. A barrier to axonal regenerative research is that the axons regenerating in response to experimental treatments stall growth before reaching post-synaptic targets. Here, we test the hypothesis that the interaction of regenerating axons with live oligodendrocytes, which were absent during developmental axon growth, contributes to stalling axonal growth. To test this hypothesis, first, we used single cell RNA-seq (scRNA-seq) and immunohistology to investigate whether post-injury born oligodendrocytes incorporate into the glial scar after optic nerve injury. Then, we administered demyelination-inducing cuprizone and stimulated axon regeneration by Pten knockdown (KD) after optic nerve crush. We found that post-injury born oligodendrocyte lineage cells incorporate into the glial scar, where they are susceptible to the demyelination diet, which reduced their presence in the glial scar. We further found that the demyelination diet enhanced Pten KD-stimulated axon regeneration and that localized cuprizone injection promoted axon regeneration. We also present a resource for comparing the gene expression of scRNA-seq-profiled normal and injured optic nerve oligodendrocyte lineage cells.

Developmentally upregulated transcriptional elongation factor a like 3 suppresses axon regeneration after optic nerve injury.

Projection neurons of the mammalian central nervous system (CNS) do not spontaneously regenerate axons which have been damaged by an injury or disease, often leaving patients with permanent disabilities that affect motor, cognitive, or sensory functions. Although several molecular targets which promote some extent of axon regeneration in animal models have been identified, the resulting recovery is very limited, and the molecular mechanisms underlying the axonal regenerative failure in the CNS are still poorly understood. One of the most studied targets for axon regeneration in the CNS is the mTOR pathway. A number of developmentally regulated genes also have been found to play a role in CNS axon regeneration. Here, we found that Transcriptional Elongation Factor A Like 3 (Tceal3), belonging to the Bex/Tceal transcriptional regulator family, which also modulates the mTOR pathway, is developmentally upregulated in retinal ganglion cell (RGCs) projection CNS neurons, and suppresses their capacity to regenerate axons after injury.

Corneal nonmyelinating Schwann cells illuminated by single-cell transcriptomics and visualized by protein biomarkers.

The cornea is the most innervated tissue in the human body. Myelinated axons upon inserting into the peripheral corneal stroma lose their myelin sheaths and continue into the central cornea wrapped by only nonmyelinating corneal Schwann cells (nm-cSCs). This anatomical organization is believed to be important for central vision. Here we employed single-cell RNA sequencing (scRNA-seq), microscopy, and transgenics to characterize these nm-cSCs of the central cornea. Using principal component analysis, uniform manifold approximation and projection, and unsupervised hierarchal cell clustering of scRNA-seq data derived from central corneal cells of male rabbits, we successfully identified several clusters representing different corneal cell types, including a unique cell cluster representing nm-cSCs. To confirm protein expression of cSC genes, we performed cross-species validation, employing corneal whole-mount immunostaining with confocal microscopy in mouse corneas. The expression of several representative proteins of nm-cSCs were validated. As the proteolipid protein 1 (PLP1) gene was also expressed in nm-cSCs, we explored the Plp1-eGFP transgenic reporter mouse line to visualize cSCs. Specific and efficient eGFP expression was observed in cSCs in adult mice of different ages. Of several putative cornea-specific SC genes identified, Dickkopf-related protein 1 was shown to be present in nm-cSCs. Taken together, our findings, for the first time, identify important insights and tools toward the study nm-cSCs in isolated tissue and adult animals. We expect that our results will advance the future study of nm-cSCs in applications of nerve repair, and provide a resource for the study of corneal sensory function.

Sex-Specific Gene Expression in the Mouse Nucleus Accumbens Before and After Cocaine Exposure.

The nucleus accumbens plays a major role in the response of mammals to cocaine. In animal models and human studies, the addictive effects of cocaine and relapse probability have been shown to be greater in females. Sex-specific differential expression of key transcripts at baseline and after prolonged withdrawal could underlie these differences. To distinguish between these possibilities, gene expression was analyzed in four groups of mice (cycling females, ovariectomized females treated with estradiol or placebo, and males) 28 days after they had received seven daily injections of saline or cocaine. As expected, sensitization to the locomotor effects of cocaine was most pronounced in the ovariectomized mice receiving estradiol, was greater in cycling females than in males, and failed to occur in ovariectomized/placebo mice. After the 28-day withdrawal period, RNA prepared from the nucleus accumbens of the individual cocaine- or saline-injected mice was subjected to RNA sequencing analysis. Baseline expression of 3% of the nucleus accumbens transcripts differed in the cycling female mice compared with the male mice. Expression of a similar number of transcripts was altered by ovariectomy or was responsive to estradiol treatment. Nucleus accumbens transcripts differentially expressed in cycling female mice withdrawn from cocaine exhibited substantial overlap with those differentially expressed in cocaine-withdrawn male mice. A small set of transcripts were similarly affected by cocaine in the placebo- or estradiol-treated ovariectomized mice. Sex and hormonal status have profound effects on RNA expression in the nucleus accumbens of naive mice. Prolonged withdrawal from cocaine alters the expression of a much smaller number of common and sex hormone-specific transcripts.

Author Correction: Single cell transcriptome profiling of retinal ganglion cells identifies cellular subtypes.

The original version of the Supplementary Information file associated with this Article contained an error in Supplementary Fig. 2. In panel c, the graph was inadvertently replaced with a duplicate of the graph in panel a. The error has now been fixed and the corrected version Supplementary Information PDF is available to download from the HTML version of the Article.

Single cell transcriptome profiling of retinal ganglion cells identifies cellular subtypes.

Retinal ganglion cells (RGCs) convey the major output of information collected from the eye to the brain. Thirty subtypes of RGCs have been identified to date. Here, we analyze 6225 RGCs (average of 5000 genes per cell) from right and left eyes by single-cell RNA-seq and classify them into 40 subtypes using clustering algorithms. We identify additional subtypes and markers, as well as transcription factors predicted to cooperate in specifying RGC subtypes. Zic1, a marker of the right eye-enriched subtype, is validated by immunostaining in situ. Runx1 and Fst, the markers of other subtypes, are validated in purified RGCs by fluorescent in situ hybridization (FISH) and immunostaining. We show the extent of gene expression variability needed for subtype segregation, and we show a hierarchy in diversification from a cell-type population to subtypes. Finally, we present a website for comparing the gene expression of RGC subtypes.

Changes in Corticotrope Gene Expression Upon Increased Expression of Peptidylglycine α-Amidating Monooxygenase.

Throughout evolution, secretion has played an essential role in the ability of organisms and single cells to survive in the face of a changing environment. Peptidylglycine α-amidating monooxygenase (PAM) is an integral membrane monooxygenase, first identified for its role in the biosynthesis of neuroendocrine peptides released by the regulated secretory pathway. PAM was subsequently identified in Chlamydomonas reinhardtii, a unicellular green alga, where it plays an essential role in constitutive secretion and in ciliogenesis. Reduced expression of C. reinhardtii PAM resulted in significant changes in secretion and ciliogenesis. Hence, a screen was performed for transcripts and proteins whose expression responded to changes in PAM levels in a mammalian corticotrope tumor cell line. The goal was to identify genes not previously known to play a role in secretion. The screen identified transcription factors, peptidyl prolyl isomerases, endosomal/lysosomal proteins, and proteins involved in tissue-specific responses to glucose and amino acid availability that had not previously been recognized as relevant to the secretory pathway. Perhaps reflecting the dependence of PAM on molecular oxygen, many PAM-responsive genes are known to be hypoxia responsive. The data highlight the extent to which the performance of the secretory pathway may be integrated into a wide diversity of signaling pathways.

Draft Genome Sequence of the Salt Water Bacterium Oceanospirillum linum ATCC 11336T.

Oceanospirillum linum ATCC 11336T is an aerobic, bipolar-tufted gammaproteobacterium first isolated in the Long Island Sound in the 1950s. This announcement offers a genome sequence for O. linum ATCC 11336T, which has a predicted genome size of 3,782,189 bp (49.13% G+C content) containing 3,540 genes and 3,361 coding sequences.

Genome Sequence of a Marine Spirillum, Oceanospirillum multiglobuliferum ATCC 33336T, Isolated from Japan.

Oceanospirillum multiglobuliferum ATCC 33336T is a motile gammaproteobacterium with bipolar tufted flagella, noted for its low salt tolerance compared to other marine spirilla. This strain was originally isolated from the putrid infusions of Crassostrea gigas near Hiroshima, Japan. This paper presents a draft genome sequence for O. multiglobuliferum ATCC 33336T.

Draft Whole-Genome Sequence of the Marine Bacterium Idiomarina zobellii KMM 231T.

Idiomarina zobellii was isolated from the northwest Pacific Ocean at a depth of 4,000 to 5,000 m in 1985. The draft whole-genome shotgun sequence of I. zobellii KMM 231(T) described in this paper has a predicted length of 2,602,160 bp, containing 2,570 total genes, 52 tRNAs, and a G+C content of 47.10%.

Genome Sequence of the Deep-Sea Bacterium Idiomarina abyssalis KMM 227T.

Idiomarina abyssalis KMM 227(T) is an aerobic flagellar gammaproteobacterium found at a depth of 4,000 to 5,000 m below sea level in the Pacific Ocean. This paper presents a draft genome sequence for I. abyssalis KMM 227(T), with a predicted composition of 2,684,812 bp (47.15% G+C content) and 2,611 genes, of which 2,508 were predicted coding sequences.