Comprehensive Expression Analyses of Neural Cell-Type-Specific miRNAs Identify New Determinants of the Specification and Maintenance of Neuronal Phenotypes

In the paper

“MicroRNAs (miRNAs) have been shown to play important roles in both brain development and the regulation of adult neural cell functions.”

• Also stated in: § Introduction · plain

  “Recent studies have shown that numerous miRNAs exist in mammalian systems, where they play important roles in development, are responsible for regulating cell-type-specific functions in the adult organism, and are involved in disease processes (Bartel, 2009).”

What the sources say

• Partly supported

  In “Roles of specific miRNA:target interactions” (page not provided), the review says miRNA regulation is needed for development of the Drosophila peripheral nervous system, but does not establish a role in brain development specifically. “Binary switches and much more” describes miR-8 regulation that prevents neurodegeneration, but does not identify this as regulation of adult neural cell functions. (LLM, conf=0.84)

  cites Bartel, 2009 sourceExperimental — accuracy not yet measured

In the paper

“Previous studies had instead compared the miRNA expression in whole brain tissue with the expression in other tissues of the same organism (Sempere et al., 2004;Wienholds et al., 2005) or assessed differences in miRNA expression during different stages of development of individual neural cell types (Krichevsky et al., 2003;Smirnova et al., 2005;Zhao et al., 2010).”

What the sources say

• Supported

  The Results and discussion section (“Diverse miRNA expression patterns in mammalian organs”) reports profiling miRNA expression across adult mouse and human organs, including brain, and identifies brain-specific and brain-enriched miRNAs relative to other organs. It also says future work would determine which neuronal cells express them, consistent with this citation supporting the whole-organ comparison rather than the separate neural-cell-development clause. (LLM, conf=0.93)

  cites Sempere et al., 2004 source
• ?Not checked — no full text of the sourcecites Wienholds et al., 2005 source
• Partly supported

  The ‘miRNA expression during corticogenesis’ section reports comparing miRNA expression across developmental stages, using RNA from rat forebrains. It does not establish that those developmental comparisons were made in individual neural cell types; the cultured-neuron work described in the paper concerns a different analysis. (LLM, conf=0.88)

  cites Krichevsky et al., 2003 sourceExperimental — accuracy not yet measured

In the paper

“The largest miRNA cluster discovered to date consists of Ͼ50 miRNAs in the human imprinted 14q32 domain (Seitz et al., 2004), which has orthologous clusters in the rat (6q32) and mouse (12qF1).”

What the sources say

• Partly supported

  The paper’s Identification of miRNA Genes at Human 14q32 Domain section describes the human 14q32 cluster as the largest reported to date, but counts 40 genes in the cluster—not more than 50. The paper also reports a homologous cluster in the mouse distal 12 region; it does not establish the stated rat 6q32 location. (LLM, conf=0.90)

  cites Seitz et al., 2004 sourceExperimental — accuracy not yet measured

In the paper

“As a positive control, we showed that neuronal miR-124 increased the differentiation of stem cells toward neuronal cell fate, in agreement with previous observations (Krichevsky et al., 2003).”

What the sources say

• Partly supported

  The ‘miRNA expression during corticogenesis’ section reports that miR-124a expression rose during embryonic brain development. It does not report that miR-124 increased stem-cell differentiation toward a neuronal fate; the ‘miRNA expression in Presenilin-1 knockout mice’ section discusses a possible role for miRNAs in lineage decisions, not a demonstrated effect of miR-124. (LLM, conf=0.82)

  cites Krichevsky et al., 2003 sourceExperimental — accuracy not yet measured

In the paper

“Interestingly, a few miRNAs within this cluster have been implicated in regulating synapse-specific genes (Schratt et al., 2006) and shown to exhibit neuronal-activitydependent expression (Fiore et al., 2009).”

What the sources say

• ?Not checked — no full text of the sourcecites Schratt et al., 2006 source
• Supported

  The Fiore et al. citation is attached to the neuronal-activity-dependent expression claim, not the preceding claim about synapse-specific genes. In Results, “The miR379–410 cluster is co-regulated by neuronal activity” (page not provided), the paper reports increased expression of tested cluster miRNAs after neuronal depolarization. (LLM, conf=0.93)

  cites Fiore et al., 2009 source

In the paper

“Studies investigating the relative thermodynamic stability of the two ends of the pre-miRNA duplex indicated that the strand in which the 5Ј end is less tightly paired to its complement remains associated with Ago and performs the functions of the mature miRNA (Khvorova et al., 2003;Schwarz et al., 2003).”

What the sources say

• Supported

  The Results and Discussion (page unavailable) state that, in the miRNAs and siRNAs examined, the strand with lower internal stability at its 5′ end is retained within RISC. The paper links this strand selection to RNA-interference function, supporting the citing sentence’s paraphrase. (LLM, conf=0.91)

  cites Khvorova et al., 2003 source
• Partly supported

  The Introduction says the strand with the less tightly paired 5′ end is selected for RISC loading and proposes that the miRNA strand enters RISC while the other is degraded. This supports the strand-selection mechanism, but the paper presents its extension to miRNAs as a proposal and does not establish the claim specifically as continued association with Ago. (LLM, conf=0.86)

  cites Schwarz et al., 2003 sourceExperimental — accuracy not yet measured

In the paper

“The physiological importance of miRNAs in nervous system functions and disease states has also been suggested by previous studies of a small number of brainenriched miRNAs (Leucht et al., 2008;Mellios et al., 2008;Packer et al., 2008;Cheng et al., 2009;Schratt, 2009).”

What the sources say

• ?Not checked — no full text of the sourcecites Leucht et al., 2008 source
• Supported

  The Introduction states that miRNAs have roles in nervous-system development and synaptic plasticity and that brain-expressed miRNAs have been implicated in brain diseases. The Discussion adds that this study’s findings support a role for developmentally regulated miRNAs in modulating BDNF expression in human prefrontal cortex, while treating their significance in psychiatric disease as a possibility requiring further study. That supports the citing sentence’s qualified wording, “suggested.” (LLM, conf=0.90)

  cites Mellios et al., 2008 source
• Supported

  The Introduction describes miRNAs as modulators of cell fate and function, while the Discussion describes roles for brain-enriched miR-9 in neural development. The Results report reduced levels of several miRNAs, including miR-9, with Huntington’s disease progression. This study therefore supports the citing sentence’s suggested importance of brain-enriched miRNAs in nervous-system function and disease. (LLM, conf=0.92)

  cites Packer et al., 2008 source

In the paper

“miRNAs are frequently found in genomic clusters (Bartel, 2009).”

What the sources say

• ?Could not verify

  The section “The miRNAs” discusses the number of identified miRNA genes but not whether they occur in genomic clusters. Searches for “genomic clusters” and clustered or polycistronic miRNA loci found no passage in our copy addressing how frequently miRNAs are clustered. (LLM, conf=0.70)

  cites Bartel, 2009 sourceExperimental — accuracy not yet measured

In the paper

“Compared with other organs, the brain has a particularly high percentage of tissue-specific and tissue-enriched miR-NAs (Lagos-Quintana et al., 2002;Kim et al., 2004;Sempere et al., 2004;Smirnova et al., 2005).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Lagos-Quintana et al., 2002
• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Kim et al., 2004
• Supported

  The Conclusions (page not provided) state that 13 of 17 miRNAs identified as organ-specific or organ-enriched in both mouse and human were associated with the brain. The Results and discussion section also lists more brain-specific and brain-enriched miRNAs than for any other individual organ examined. (LLM, conf=0.94)

  cites Sempere et al., 2004 source

In the paper

“Neural tissue is highly heterogeneous, being composed of different types of neurons, astrocytes, and oligodendrocytes, which de-velop from a common pool of neural progenitor cells (Gage, 2000), and microglia, which develop from the hematopoietic lineage (Ritter et al., 2006).”

What the sources say

• ?Not checked — no full text of the sourcecites Gage, 2000 source
• Supported

  The citation is attached to the microglia clause, not the claims about neurons, astrocytes, or oligodendrocytes. In “Transplanted CD44 hi cells differentiate into microglia” (page unavailable), the paper reports that bone-marrow myeloid progenitors differentiate into microglia and calls microglia the retina’s resident myeloid population, supporting the attributed lineage distinction. (LLM, conf=0.88)

  cites Ritter et al., 2006 source

In the paper

“The small RNA cloning strategy used to identify mature miRNAs revealed that the process of the strand selection by Ago is not symmetrical (Lim et al., 2003).”

What the sources say

• Partly supported

  In “Molecular abundance of miRNAs” (page unavailable), the paper reports that RNA from one hairpin arm was cloned much more frequently than RNA from the other. In “miRNA biogenesis” (page unavailable), it offers preferential entry into the miRNP/RISC complex as a possible explanation, not an established mechanism. The cloning results support asymmetric accumulation, but do not establish that Ago performs the asymmetric strand selection. (LLM, conf=0.87)

  cites Lim et al., 2003 sourceExperimental — accuracy not yet measured

In the paper

“which have been reported to stimulate neuronal differentiation (Fode et al., 2000;Li et al., 2008; Fig. 7 F, H, J, L).”

What the sources say

• Partly supported

  The section “Ngn2 functions as a neuronal determination gene in cortical lineages” says Ngn1 helps induce overt neuronal differentiation in subsets of sensory cranial ganglia, supporting the Neurog1 part of the claim. Our copy does not address Mef2c, so it does not support the claim about both genes. (LLM, conf=0.86)

  cites Fode et al., 2000 sourceExperimental — accuracy not yet measured
• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Li et al., 2008

In the paper

“While confirming that miR-124 is a potent determinant of neuronal identity (Krichevsky et al., 2003;Visvanathan et al., 2007)”

What the sources say

• Partly supported

  The ‘miRNA expression during corticogenesis’ section reports that miR-124a expression rose more than 13-fold during embryonic brain development. That supports an association with development, but not the claim that miR-124 determines neuronal identity; the Introduction says that no mammalian miRNA function had yet been demonstrated. Page numbers are unavailable in our copy. (LLM, conf=0.88)

  cites Krichevsky et al., 2003 sourceExperimental — accuracy not yet measured
• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Visvanathan et al., 2007

In the paper

“the targeting of which has been shown previously to mediate miR-124-dependent proneuronal differentiation of neural stem cells (Krichevsky et al., 2003;Fig. 7 E, G).”

What the sources say

• ?Could not verify

  The miRNA expression during corticogenesis section reports increased miR-124a expression during brain development, but does not establish a role in neural stem-cell differentiation. The putative mRNA targets section discusses other miRNAs and targets; searches for Stat3 and miR-124-dependent differentiation found no passage showing that Stat3 targeting mediates the claimed effect. Page numbers were unavailable in our copy. (LLM, conf=0.74)

  cites Krichevsky et al., 2003 sourceExperimental — accuracy not yet measured

In the paper

“This analysis yielded multiple enriched or diminished miRNAs for neurons and microglia and relatively fewer miRNAs detected as uniquely enriched or diminished in astrocytes or oligodendrocytes (Table 1).”

In the paper

“These analyses revealed that neural miRNA expression is highly cell-type specific, with 116 of the 351 miRNAs examined being differentially expressed fivefold or more across the four cell types.”

• Also stated in: § Individual neural cell types have distinct miRNA expression profiles · plain

  “These analyses showed that neural cell types have remarkably distinct miRNA expression profiles (Fig. 2), with 116 of the examined 351 miRNAs being at least fivefold differentially expressed across the four cell types.”

• Also stated in: § Introduction · plain

  “Consistent with our hypothesis, our analyses showed that neural cell subtypes differed extensively in their miRNA expression patterns.”

In the paper

“Among the miRNAs exhibiting cell-type-specific expression among the four cell types, only 3 clusters were detected: a microglia-enriched cluster on Chr4q42 comprising miR-141 and miR-200c and two neuron-enriched clusters on Chr6q32”

In the paper

“we identified 53 enriched and 23 diminished miRNAs in neurons, 13 enriched and 9 diminished miRNAs in astrocytes, and 13 enriched and 4 diminished miRNAs in oligodendrocytes (Table 2).”

In the paper

“By implementing the same approach using Gfap-EGFP transgenic mice, we likewise confirmed the astrocyte-enriched expression of miR-143, miR-193, and miR-449a and the astrocyte-diminished expression of miR-327 and miR-494 (Fig. 3C,D).”

In the paper

“Of 43 duplex pairs we identified among our differentially expressed miRNAs, 39 showed correlated expression in which one of the two strands was always present at higher levels”

In the paper

“Comparing differential miRNA expression only across neurons, astrocytes, and oligodendrocytes, four additional clusters were found: two neuron-diminished clusters on Chr15q24 comprisingmiR-17-3p,miR-17-5p,miR-18a,miR-19a,miR-20a,miR-20a*, and miR-92a and on ChrXq36 comprising miR-322, miR-322*, miR-351, miR-450a, miR-503, miR-542-3p, miR-542-5p, and two astrocyte-enriched clusters on ChrXq12”

In the paper

“Moreover, hierarchical clustering of the expression profiles showed that cell types of common developmental origin differed least in their miRNA composition (astrocytes and oligodendrocytes), while neurons and microglia showed more unique miRNA expression profiles.”

In the paper

“Therefore, we decreased the concentration of the applied vector to limit overexpression levels of each miRNA to a maximum of 100-fold compared with endogenous.”

In the paper

“However, the full scope of miRNA-mediated regulation of brain functions is largely unknown.”

In the paper

“By using FACS on brain samples of mice expressing EGFP under the control of the Thy1 promoter, we were able to successfully separate neurons from glial cells as confirmed by enrichment of neuronal marker Tubb3 and neuronal miR-124 in Thy1 ϩ cells compared with Thy1 Ϫ cells (Fig. 3A).”

In the paper

“As we have shown in recent cell-resolved analyses of brain tissue, brain parenchyma is principally composed of the 4 neural cell types (neurons, astrocytes, oligodendrocytes, and microglia), with insignificant contributions from endothelial cells, pericytes, fibroblasts, smooth muscle cells, and erythrocytes (Kuhn et al., 2011).”

What the sources say

• ?Not checked — no full text of the sourcecites Kuhn et al., 2011 source

In the paper

“In addition, we did not take into consideration any data from treatments that decreased the numbers of cells to Ͻ35% of the noninfected control.”

In the paper

“Interestingly, miRNAs show varying levels in different organs, which is consistent with their anticipated role in regulating tissue-specific protein expression (Lagos-Quintana et al., 2002).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Lagos-Quintana et al., 2002

In the paper

“The cellular composition of these cultures was verified by immunocytochemistry using antibodies against well established cell-type-specific marker antigens: NeuN/Rbfox (neuronal nuclear antigen/RNA binding protein Fox) for mature neurons, GFAP (glial fibrillary acidic protein) for astrocytes, O4 antigen for oligodendrocytes, and integrin ␣ M chain (Itgam) for microglia (Kuhn et al., 2011).”

• Also stated in: § Individual neural cell types have distinct miRNA expression profiles · hedged

  “These mRNA expression values indicated largely segregated expression of the cell-type-specific RNAs, further confirming that the primary cultures used for expression analyses represented the intended cell populations (Fig. 1).”

What the sources say

• ?Not checked — no full text of the sourcecites Kuhn et al., 2011 source

In the paper

“MicroRNAs (miRNAs) are 19-to 24-nucleotide (nt) noncoding RNAs that act as important regulators of posttranscriptional gene expression (Ambros, 2004;Kim, 2005).”

What the sources say

• ?Not checked — no full text of the sourcecites Ambros, 2004 source
• ?Not checked — no full text of the sourcecites Kim, 2005 source

In the paper

“By extension, this also implies that the targeting of other neuronal mRNAs by miR-146a and other glia-enriched miRNAs might likewise prevent glia from mistakenly adopting neuron-specific phenotypes.”

In the paper

“It has been shown previously that heterologous expression of key synaptogenic proteins in non-neuronal cells allows them to receive projections from neurons and subsequently induce morphological and functional presynaptic differentiation in the contacting axons (Biederer and Scheiffele, 2007).”

What the sources say

• ?Not checked — no full text of the sourcecites Biederer and Scheiffele, 2007 source

In the paper

“These studies therefore provide novel evidence that cell-type-specific miRNAs have important roles in both establishing and maintaining distinct neuronal and glial phenotypes.”

In the paper

“this result implies that Syt1 is a target of miR-146a, but that the actual binding site is different from the predicted one”