Long Non-Coding RNA in Neural Stem Cells Self-Renewal, Neurogenesis, Gliogenesis and Synaptogenesis

Abstract

Evidence reports the key roles of lncRNAs in several regulatory mechanisms of neurons and other brain cells. Neuronal lncRNAs are crucial for NSCs mediated-neuronal developmental stages like neurogenesis, neuronal differentiation, and synaptogenesis. Moreover, multilineage properties of NSCs and their association to specific cell types render them to identify the commonly accepted biomarkers for the brain. It is important to delineate the correlation between lncRNAs and NSCs fate decisions during neuronal development stages. In this review, we will summarize how NSCs fabricate embryonic tissue architecture of the central nervous system (CNS) and act as residuum in subventricular zone (SVZ) nearby the lateral wall of the lateral ventricles and the subgranular zone (SGZ) of hippocampus dentate gyrus (DG) of the adult brain. Additionally, describe the roles and molecular mechanisms of lncRNAs involved in NSCs self-renewal, neurogenesis, gliogenesis and synaptogenesis over the course of neural development. This will help us to better understand neuronal physiology.

1. Introduction

In the central nervous system (CNS), an efficient spatio-temporal regulatory mechanisms play an important role in neural stem cells (NSCs) for the development of neurons and other brain cells. The development and differentiation of the CNS is intricate in nature, and plethora of regulatory factors are involved and express ~40% of lncRNAs [1]. lncRNAs are involved in the early development/differentiation of the nervous system (NS) through NSCs to synaptogenesis [2, 3, 4]. Field et al. [5] constituted pluripotent stem cell (PSC)-derived cerebral cortex organoid (CO) cell cultures which recapitulate the cellular organization and gene expression events observed in fetal tissue. PAX6 (neural progenitors), CTIP2 and TBR1 express during early deep-layer neuron and TBR2 expression is in intermediate progenitors in Human COs at day 35. The ratio of human COs, pluripotency markers such as OCT3/4 were down-regulated, while early neural stem cell markers, including PAX6, were up-regulated during the first week. Subsequently by 5th week TBR1 were strongly expressed in Deep-layer neurons. Gene Ontology (GO) term analysis showed significant enrichment of lncRNA associated with neuronal development along with prefrontal cortex and foetus brain as described in Table 1. On assessing lncRNAs, previously described mammalian conserved lncRNAs MALAT1, NEAT1, H19, PRWN1, and CRNDE and 79 unannotated lncRNAs were observed among the 920 primate-conserved category. Markedly, in week 2 transient expression of lncRNAs (TREX) originated from TREX2174 (RP11-314P15) which includes 19 bp insertion overlay at its transcription start site. Importantly, TREX4039 (extends over AC011306 and MIR217HG) peaks at first or second week human COs and disappears by fifth week. At week 2 of human Cos gene expression specific single cells were processed for scRNA-seq libraries and TREX, which have potential roles in early cortical cell fate specifications were identified as depicted in Table 2. The clusters identified at week 2 (neuroepithelium (NE) cells-cluster of 1261 cells; early-forming Cajal-Retzius (CR) cells-cluster of 356 cells; the cortical radial glia (RG)-cluster of up 2593 cells were converted into clusters of early neurons (26%), intermediate progenitors (11%), mature radial glial (RG) (26%), immature RG (18%), dividing RG (12%), and cell doublets (8%) at week 5 of organoid cells.

Human cell atlas		ARCHS4
Week 2-GO-Term enrichment	Week 5-GO-Term enrichment	Week 2-GO-Term enrichment	Week 5-GO-Term enrichment
Fetal brain	Fetal brain	Motor neuron	Motor neuron
Prefrontal cortex	Prefrontal cortex	Prefrontal cortex	Prefrontal cortex
Amygdala	Amygdala	Neuronal epithelium	Spinal cord
Pineal night	Pineal night	Cerebellum	Spinal cord (bulk)
Pineal day	Pineal day	Spinal cord	Cerebellum
Smooth muscle	Cerebellum peduncle	Spinal cord (bulk)	Fetal brain
Occipital lobe	Whole brain	Mid brain	Cingulate gyrus
Uterus	Cardiac myocytes	Fetal brain cortex	Sensory neuron
Prostrate	Cerebellum	Fetal brain	Cerebral cortex
Cardiac myocytes	Occipital lobe	Sensory neuron	Brain (bulk)

Table 1.

The top 10 enriched GO terms from ARCHS4 [6]; based on publicly available RNA-seq data from human and mouse) and Human Cell Atlas ([7]; based on microarrays of human and mouse tissues) ranked by their combined enrichment score [5].

Name of cells	Gene expression markers	Cluster of single cells	Enrichment of transiently expressed lncRNAs (TREX) at week 2	Activity of lncRNA in trans manner
Neuroepithelium (NE) cells	HES3 and NR2F1	Cluster of 1261 cells	TREX108 and TREX8168	Genes associated with whole brain, superior frontal gyrus, and cerebral cortex suggesting a role in general neural gene networks
Cajal-Retzius (CR) cells	TBR1, EOMES, LHX9, and NHLH1	Cluster of 356 cells	TREX4039	Induced genes enriched in the ARCHS4 neural epithelium gene set and repressed expression of those associated with superior frontal gyrus and astrocytes
Cortical radial glia (RG)	SOX2, EMX2, NNAT, PTN, and TLE4	Cluster of 2593cells	TREX5008	Induced genes enriched in the ARCHS4 neural epithelium gene set and repressed expression of those associated with superior frontal gyrus and astrocytes

Table 2.

10× Chromium 3′ end scRNA-seq on week 2 human COs gene expression based neuroepithelium (NE) cells, were identified by expression of HES3 and NR2F1, forming a cluster of 1261 cells.

Early-forming Cajal-Retzius (CR) cells expressed TBR1, EOMES, LHX9, and NHLH1, comprising a cluster of 356 cells. The largest cluster strongly expressed cortical radial glia (RG) markers SOX2, EMX2, NNAT, PTN, and TLE4, making up 2593 cells showed discovery of transiently expressed lncRNAs, which have potential roles in early cortical cell fate specifications [5].

The study showed the importance of cellular specificity of lncRNA function [8]; robust regulatory effects on distal genes upon activation/repression of these TREX lncRNAs was also observed (Table 2). In our recent review [9], we have already discussed the mechanisms to identify lncRNA, synthesize lncRNA, transcription of lncRNA and localized processing of lncRNA in nuclear and cellular portions, and their regulatory functions mediated by linking to chromatin alone or by constructing lncRNA-protein-chromatin complexes owing to chromatin modifications and genomic stability, hence influencing pluripotency. LncRNA are also involved in transcriptional and post transcriptional regulation and serve as scaffolds, chromatin modifiers and miRNA sponges have also been explained in our recent review [9]. This review aims to explain the roles and molecular mechanisms of lncRNAs focusing on NSCs self-renewal, neurogenesis, gliogenesis and synaptic excitability over the course of neural development.

2. Role of NSCs in designing embryonic tissue architectonics of the central nervous system

Embryonically, the neurons of the brain are organized into layers (cortices) and clusters (nuclei), each having different function and interrelation with other neurons. The embryonic neural tube comprises single cell layer thick germinal neuroepithelium (GNE) which encompasses most proliferative neural stem cells. The GNE continues from the outer edge to the lumen of the neural tube [10], the nuclei of GNE exist at discrete levels, hence giving the impression of numerous cell layers in the neural tube. The nuclei move inside GNE as they move forward to different G0/G1, S and M phases of cell cycle. First and foremost, the S phase or DNA synthesis occurs when the nucleus is at the outer border of the neural tube, subsequently the nucleus moves towards lumen of the neural tube as the cell cycle proceeds.

However, after a certain time period some of the GNE-cells stop DNA synthesis and mitosis and migrate and differentiate into neuronal and glial cells outside the neural tube [11, 12]. The dividing cells of GNE have been found in the outer cortex of the adult brain, this was explained by radioactive thymidine studies. The published reports suggested neuroepithelial stem cells split “vertically” instead of “horizontally.” After vertical division, the daughter cell besides the lumen of the neural tube remains affixed to the ventricular surface (and persists as stem cells in the ventricular zone), while the other daughter cell migrates away [13]. This ultimate vertical division is the terminal division and origination of a neuron happens. The cells which originate late move through GNE and form the most superficial regions of the cortex. Consecutive differentiation is based on the inhabitancy of the new neurons once outside the GNE [14, 15]. The process of division persists i.e., the cells next to the lumen sustain division and are exported outwards to the lumen. In such a manner they form a second layer and sequential addition of more cells derived from GNE thickens and widens the outermost layer and forms mantle (or intermediate zone). The GNE itself acts as a ventricular zone (later known as ependyma that embraces neural stem cells). Further, the cells of the mantle zone differentiates both in neurons and glia. The axons of neurons make connections and move away from the lumen while glial cells form sheath around axons and these are suggested as white matter. While the neuronal cell bodies represent the grey matter.

3. Spinal cord and medulla assembly

In the spinal cord and medulla, three-zone decorum first-ependymal, second-mantle, and third-marginal layers are maintained throughout development. The grey matter (mantle) is a butterfly-shaped structure and is surrounded by white matter; both grey and white matter are enclosed by the connective tissue. Further, on maturation the neural tube is divided into dorsal and ventral halves through a longitudinal groove, the sulcus limitans. The dorsal portion receives input from sensory neurons, whereas the ventral portion is intricated in performing various motor functions.

4. Cerebellar structure

In cerebellum the different neurons which migrate outside the GNE through cell migration, further undergoes differential neuronal proliferation, and selective cell death that creates modifications in the three-zone pattern (ependymal, mantle, and marginal layers).

In the cerebellum, a small quantity of neuronal precursors i.e., neuroblasts derived from GNE either enter the marginal zone to form clusters of neurons called nuclei or migrate to the outer surface of the neural tube to form a new germinal zone. Each single nucleus of the marginal zone works as an individual functional unit, and connects to the outer layers of the cerebellum and other parts of the brain. The new germinal zone forms the outer layer of the developing cerebellum and is also known as the external granule layer-EGL (two cells thick layer. The outer layer of the EGL is composed of dividing neuroblasts, while the inner layer of EGL comprises postmitotic neuroblasts. The latter are the precursors of the preeminent neurons of the cerebellar cortex and the granule neurons. Granule neurons are the major component of the internal granule layer-IGL that migrates back to white matter of developing cerebellum. Concurrently, the ependymal layer (ventricular zone of GNE) differentiates into neurons and glial cells which include the unique Purkinje neurons. Purkinje neurons interact to form electrical synapses (unlike chemical synapses, there is direct interaction between presynaptic and postsynaptic neurons and also support granule neurons. These neurons secrete a specific morphogen Sonic hedgehog (SHH), which maintains the division of granule neuron precursors (neuroblasts) in the EGL [16].

Each Purkinje neuronal cell body is shaped like a flask and has a large, flat, highly branched thread like extensions creating a dendritic Arbor. This helps in forming hundreds of thousands connections (synapses) with other cells like Bergmann glial cells and granule cells. The axon of each Purkinje neuron connects to neurons in the deep cerebellar nuclei and transmits impulses to the region of cerebellum which controls movement.

The evolution of spatial arrangement of neurons is an essential process for the development and functioning of the cerebellum. Positioning of young neurons is under glial guidance [17, 18]. Importantly, the granule cell precursors (neuroblasts) move forward through adhesion on the long axonal processes of the Bergmann glia [19, 20]. The neural-glial reciprocal interaction between glial cells and neuroblasts [17, 21] is held up by adhesion protein called astrotactin that assists in adhesion of neurons to the glial cells [22, 23].

5. Cerebral structure

The three-zone positioning pattern cerebellum first-ependymal, second-mantle, and third-marginal layers of the neural tube is modified to vertical and horizontal positioning in the cerebrum.

Vertical positioning shows different vertical layers which interact with one another. As in cerebellum, in cerebrum, some of the neuroblasts move outwards from GNE and form the mantle zone and travel under glial guidance along the white matter to produce an additional layer of neurons at the outer layer of the cerebral cortex. This additional layer of grey matter (mantle) is called the neocortex. Neocortex is 2–5 mm thick, present on the surface of brain time-dependently differentiates into six layers (molecular layer, external granular layer, external pyramidal layer, internal granular layer, ganglionic layer and multiform layer) of neuronal cell bodies. Although layers are premature in nature and the maturity is not completed until the middle of childhood. Each vertical layer of the neocortex is unique and holds a specific function based on the types of neurons, and their connections with other neurons. For example, the inputs sent by thalamus are received by the neurons of layer 4, while output back to the thalamus is sent by the neurons of layer 6.

Horizontal positioning of the layers in cerebrum is more complex and they approximately form almost 40 regions that are regulated physiologically and have specific functions. For example, neurons in vertical cortical layer 6 horizontally possess both “visual cortex” and “auditory cortex”. Auditory cortex is more anterior in position than the visual cortex. Visual cortex projects axons to the lateral geniculate nucleus of the thalamus (for vision), while auditory cortex projects axons to the medial geniculate nucleus of the thalamus (for hearing).

The vertical and horizontal arrangement in cerebrum depends on numerous multilineage neuroblasts derived from GNE. Further, on terminal mitotic division most of the neuroblasts of ventricular (ependymal) progress under glial guidance and lead to the formation of cortical plates located at the outer surface of the cortex of cerebrum.

To conclude, the neuroblasts with the early origin derived from GNE form the layer adjacent to the ventricle while neurons of later origin migrate through GNE and set apart to form the more exterior layers of the cortex. This positioning of cells forms an “inside-out” descent of expansion [24]. The neuroblast of the ventricular (ependymal) zone divides into neurons and glial cells in any of the cortical layers [25]. However, the fates of neuroblasts depend on the terminal division. The neuroblasts early in development are in their mid-S phase i.e., are in the way of division i.e., final division is not complete, hence likely become any neuron (for instance neurons of layers 2 or 6), at later stage of development of neuroblasts final mitosis is complete that give rise only to upper-level (layer 2) neurons [26]. The migration of the majority of these young neurons occurs in a radial manner on glial processes [27]. The migration initiates from the ventricular zone to the cortical plate. After neurogenesis, the generated young neurons (~12%) migrate laterally from one region of the cerebral cortex into another [28]. Further, the positioning of young neurons in different cortical areas are designated to unique functional domains. Once the neuronal cells make an appearance at their final designated cortical area, they may produce specific adhesion molecules that sort them together as brain nuclei [29].

6. Role of adult neural stem cells (NSCs) in neurogenesis

The cortical regions are composed of abundant neurons and glial cells produced from the differentiation of GNE-derived neuronal precursor cells/neuroblasts, which are self-sufficient to renew themselves as well as differentiate into multilineage neurons in the brain [30]. The above process of producing neurons and glial cells are termed neurogenesis and gliogenesis, respectively [31]. The largest NSCs/neuronal precursor cells/neuroblasts niches all through the life are essentially located in the adult ventricular-subventricular zone (V-SVZ) adjacent to the walls of the lateral ventricles [32] and the subgranular zone (SGZ) in the dentate gyrus of the hippocampus where new dentate granule cells are produced.

6.1 NSC model in the adult subventricular zone under basal conditions

V-SVZ produces large number of neuroblasts which travel a long distance through the rostral migratory stream (RMS) to the olfactory bulb (OB) in form of a chain, where they finally differentiate into granule cell type local interneurons [33]. Further, once reaching the core of the OB, immature neurons separate from the RMS through radial migration and then migrate towards glomeruli and differentiate into various subtypes of periglomerular (PG) interneurons. PG interneurons interconnect with the apical dendrites (often lack axons) of mitral and tufted cells inside the glomeruli [34]. The majority of interneurons are GABAergic granule cell neurons, which lack axons and form dendro-dendritic synapses with lateral dendrites of mitral and tufted cells in the external plexiform layer. In addition, few number of GABAergic PG interneurons and small percentage of interneurons are dopaminergic in nature. One report also suggested that very low percent of new interneurons are glutamatergic juxtaglomerular neurons in nature [35]. Transcript based markers have been identified for the five developmental stages of adult SVZ neurogenesis of the lateral ventricle and OB [1] activation of radial glia-like cells (GFAP, Vimentin and nestin positive) in the SVZ zone in the lateral ventricle (LV); [2] proliferation of transient amplifying cells/progenitor cells (Mash1 and low amount of nestin); [3] produce neuroblasts (Dlx2 positive) that migrate to OB; [4] subsequent chain migration of neuroblasts (Dlx2 positive) within the RMS and radial migration of immature neurons (Dlx2 and DCX positive) in the OB; [5] Synaptic interaction through interneurons (NeuN positive) and maturation of GC and PG neurons in the OB (reviewed by [33]) .

6.2 NSC model in the adult hippocampus under basal conditions

Adult SGZ in the dentate gyrus of the hippocampus possess proliferating radial and non-radial neuronal precursor cells that generate intermediate progenitors/transit amplifying cells, which in succession generate neuroblasts.

The multi-lineage radial precursors i.e., radial glia-like cells (RGLs or Type-1 cells) in dentate gyrus of adult hippocampus are identified through expression of transcripts like nestin, GFAP and Sox2. Besides transcript markers a distinguishing feature RGL possesses is radial branch which extends through the granule cell layer. Single RGL undergoes several rounds of self-renewal and differentiation to produce both neurons and astrocytes for a long period, displaying characteristic stem cell properties by each RGLs as evidenced through in vivo clonal assays [36].

Quiescent RGLs once triggered from quiescence stage i.e. G0 phase, RGLs moves towards formation of specific cell types like RGLs, non-radial precursors, proliferative intermediate progenitors (IPCs, or Type-2 cells) and astroglia, excluding the oligodendrocyte lineage. Later on the fate of RGLs may be decided whether they remain in a proliferative state, return to quiescence, or differentiate into an astrocyte.

Non-radial precursors generate new neurons in the adult SGZ and act as primary precursors [37]. They lack any radial process and few cells present as parallel extensions to the dentate granule cell layer. Unlike RGL, non-radial precursors express Sox2, but not GFAP [38] and more proliferative i.e., mostly present in cell cycle [37, 38]. They may or may not show involvement of IPCs and may generate new RGLs or astroglia, while maintaining the precursor state [36].

IPCs of the SGZ region of dentate gyrus rapidly proliferate and possess small tangential processes expressing Tbr2 as transcript marker [39]. They are derived from both radial and non-radial precursors [36, 38]. Afterwards they convert into secondary transient amplifying precursors and start expressing DCX and Prox1, markers of committed immature neurons [39]. IPCs re-enter cell cycle for self-renewal or produce astroglia or maintain as precursors over a long duration remains unclear.

In adult dentate non-neurogenic areas and SGZ region enrichment of another, progenitor population of NG2 cells have been identified [40] Major features of NG2 cells are long wispy processes, expression of specific proteogylcan NG2 as well as PDGF, Sox10 and Olig2 [41] has been observed. Presence of NG2 cells and oligodendrocytes in non-neurogenic areas differentiate them from RGLs in the adult SGZ [36] suggesting that they may have derived from different precursor populations with different embryonic origins. The consensus remains that NG2 cells may produce astroglia during development, and oligodendrocyte generation in the adult nervous system [41].

Astroglia, non-neuronal quiescent precursor cell type as proposed by absence of nestin expression, are a prospective third precursor population in the adult SGZ. Major characteristic features are horizontal or bushy morphology and expression of GFAP, S100β and Aldh1l1 markers [42]. The new astroglia generate from RGLs in the adult SGZ; afterwards they migrate to the hilus or molecular layer [36]. Hence, neuroblasts from NSCs in SGZ migrate short distances into the granule cell layer and mature into neurons, then integrate into functional circuits [43].

7. The effect of lncRNA on NSCs/NPCs pluripotency

Cell type- and tissue-specific long non-coding RNAs (lncRNAs) incorporate varying classes of transcripts that can control various fundamental molecular and cellular processes in organ development, disease and cancer. Here we will discuss the differential expression and unique function of lncRNAs in human brain development.

LncRNAs are present amply and specifically in different lineages of neurogenic cell-types which plays an important role in neuronal development [8]. For instance, Liu et al. [44] identified cell type-specific lncRNA and mRNA transcript pairs in developing neocortex; (a) radial glia-specific lncRNA LOC646329-mRNA PAX6, (b) maturing neuron-specific lncRNA LINC00599-mRNA RTN1, (c) interneuron-specific lncRNA DLX6-AS1-progenitor and differentiated cell-expressed mRNA NNAT. Maturing neurons of the cortical plate (CP) were enriched with LINC00599. The subpial granular layer interneuron showed predominance of unique DLX6-AS1 [45, 46].

8. The function of lncRNAs in neurogenesis/neural differentiation

LncRNAs play a pivotal role in regulating epigenetic elements in NPCs or NSCs differentiation and neural development [47, 48]. Spatial and temporal expression of lncRNAs plays a very crucial role in neuronal development in the developing CNS [49]. However, the abundance and specificity of lncRNAs in different neurogenic cell-types or the specific functions show their involvement in development and cellular identity in the nervous system [1, 8].

Recently, a gene expression atlas of embryonic neurogenesis in Drosophila revealed complex spatiotemporal regulation of lncRNAs. This involved a significant set of 13 lncRNAs, on illustration they represented [1] time dependent appearance at subcellular and cellular levels, [2] moderate to excess expression in cytoplasmic or nuclear regions [3] were involved in precise RNA processing (for example splicing and nuclear-cytoplasmic commutation), during critical events in neurogenesis in Drosphila Table 3 [50].

CR30009	CR30009 is spliced and primarily exported to the cytoplasm	Increased expression in the early IC and in NBs (4–6 h and 6–8 h) constitutes the earliest neuroblast marker of the glial lineage; However, was most highly enriched in glial cells stage 9/10 and stage 13/14	Specific expression in NBs and shows increased expression in glial cells
CR43283 (also known as cherub)	Specifically localized to the cytoplasm throughout embryogenesis and is clearly spliced, but harbors no coding potential and showed dynamic temporal regulation	Expression of cherub was strongly enriched in the earliest neuroblasts at 4–6 h, but enrichment quickly decreased in later neuroblasts (6–8 h); however, over time cherub became specifically expressed being strongly enriched in differentiated neurons and glia by the end of neurogenesis at 18–22 h	Specific expression in NBs and shows increased expression in differentiated neurons and glia
CR32730	Moderately enriched in the nuclear fraction in early and late embryos	CR32730 first detected in 4-6 h neuroblasts and was moderately enriched at 8–10 h in the neuronal, but not in the glial, population	Specific expression in NBs and shows increased expression in early neurons
CR46003	CR46003 was one of the most abundant and did not exhibit clear subcellular enrichment in either early or late embryos	First detected in the ventral column and was most highly enriched in early neuroblasts, but expression persisted in neuroblasts and early neurons	Specific expression in NBs and shows increased expression in early neurons
CR44024	Not predicted to exhibit distinct subcellular localization in early (6-8 h) embryos, but was moderately enriched in the cytoplasm at the end of embryogenesis (18–22 h)	Was first enriched in early neuroblasts and persisted through neuronal differentiation, and is predicted to be excluded from the intermediate and ventral columns and glia	Specific expression in NBs and persistent expression in neurons

Table 3.

A list of high-confidence set of lncRNAs classified time dependently; distinct subcellular localization patterns and cell-specifically; moderate to overabundance in cytoplasmic or nuclear regions; and involved in highly specific RNA processing in neurogenesis in Drosphila.

Biological materials studied over the time course of neurogenesis: intermediate column (IC); ventral column (VC); neuroblasts (NBs); neurons; glia. Markers specifically used for RNA-FISH was NBs, Pros; neurons, Elav; glia, Repo (4–6 and 6–8 h (IC, VC, NBs); 6–8, 8–10 and 18–22 h (neurons and glia)) [50].

Interestingly, lncRNAs have undergone specific adaptive functions like selective loss during the evolution of neurogenesis [51]. LncRNA was a key determinant in NSCs or NPCs during cell-fate determination. Additionally, specific lncRNA types are involved in the different stages of NPCs or NSCs differentiation. The neuronal and astrocytic differentiation have been well explained based on a plethora of differentially expressed epigenetically modified lncRNA. It has also been suggested that different lncRNAs have biasness for neuronal differentiation compared to astrocytic differentiation. Although astrocytic differentiation is related more to the sense lncRNAs (lncRNA transcribed from the sense strand of exons possessing coding genes) [52]. Besides, the above functions lncRNAs also participate in synchronizing the fate of NSC differentiation into glia and neurons under physiological and pathological conditions [53, 54]. An overview of lncRNA involved in neurogenesis/neuronal differentiation, gliogenesis or synaptogenesis has been well described in Table 4.

LncRNA name	Mechanism	Biological function	References
Sox2ot	CpG island of Sox2, interacts with transcription factor YY1 and suppresses the expression of Sox2	Prohibit NSCs proliferation and advance neuronal differentiation	[55]
RMST	Target Sox2 promoter region	Promote neurogenesis	[56]
Kdm2b (also known as Kancr)	Bind with hnRNPAB and activate Kdm2b gene expression	Causes early neuronal differentiation of cortical projection neurons, hence promotes neurogenesis	[57]
Paupar	Bind with local epi-regulatorygenes-Pax6 and KAP1 through H3K9me3 deposition	Promote neurogenesis in neuroblastoma cells	[58, 59]
Gm21284	Interact with miR-30e-3p, miR-431 and miR-147	Inhibit NSCs proliferation while promote NSCs differentiation	[60]
1604	miR-200c/ZEB1/2 axis	Promote neural differentiation	[61]
Rik-201	Activated by C/EBPβ, miR-96/Sox6	Enhance neural differentiation	[62]
Rik-203	miR-467a-3p/Sox6, miR-101-3a/GSK-3β	Enhance neural differentiation	[62, 63]
MEG3	Act as a negative regulator of miR-128-3p while induced by the cAMP/response element-binding protein (CREB) pathway	Promotes neuron differentiation	[64]
Malat1	Activate ERK/MAPK, inhibit PPAR/p53	Promote neural differentiation in neuroblastoma-derived Neuro-2a (N2a) cell	[65]
Pnky	Interact with RNA-binding protein (RBP)-PTBP1	Inhibit neural differentiation and neurogenesis	[30, 66]
lncR492	Interact with HuR and activate Wnt signalling	Inhibit neural differentiation of mouse embryonic stem cells	[67]
BDNF-AS	Targeting activating potassium uptake system protein (TrkB) signaling pathway	Inhibit eNSCs-derived neurite outgrowth and neural apoptosis	[68]
UCA1	miR-1 and its target Hes1	Promote NSCs differentiation to astrocyte not to neuron	[54]
lnOPC	lnOPC binds to upstream sequences of OLIG2	Promotes OPCs differentiation and oligodendrogenesis	[69]
lncOL1	Form a complex with Suz12, an oligodendrocyte maturation promoter	Promote oligodendrogenesis by promoting early maturation/differentiation of oligodendrocytes in neural development	[70]
lnc158	Promote regulatory transcription factor-nuclear factor-IB NFIB expressio	Promote oligodendrogenesis through enhanced oligodendrocyte-related genes expressions like and enhanced induction of oligodendrocyte lineage differentiation	[71]
Pcdh17it (immature lncRNa)		Oligodendrogenesis marker identified in new-born immature OLs	[72]
OLMALIN/-AS	OLMALINCAS, maps to the first exon of the dominant isoform of OLMALINC	Regulate oligodendrocyte maturation related genes	[73]
Synage (includes three isoforms of Gm2694)	Synage as a sponge for the sponge to microRNA miR-325-3p, act as scaffold for organizing the assembly of the LRP1-HSP90AA1-PSD-95 complex	Regulating synaptic stability in cerebella as distributed in the cytoplasm and synapses of cerebellar cells	[74]
GM12371	Nuclear-enriched	Prolific transcriptional regulator critical for synapse function in hippocampal neurons	[75]
Gm2694 (alias AK082312)	Enriched expression in the mouse cerebellar cortex		[76]
Gm2694; lncRNA (alias linc1582)		Associated with neuroectoderm differentiation	[77]
Gm2694; (alias Trincr1) was documented to	Regulate FGF/ERK signaling	Self-renewal of NSCs	[78]

Table 4.

The roles of lncRNAs on NSCs differentiation/neurogenesis, oligodendrogenesis and synapse stability.

9. The function of lncRNAs in neuronal differentiation

LncRNAs are cardinal for neuronal differentiation and for neurogenesis. They are specific to the brain region, especially SVZ, DG or Olfactory Bulb (OB). They exert their functions via interacting with transcription factors or binding to the promoter or enhancer regions of neighbouring/target genes, also act as competing endogenous RNA (ceRNA) against synergistic binding sequences of miRNA or are crucial signaling pathway modulators which regulate chromatin modification, transcription, and post-transcription.

10. Effect on lncRNA target genes expression

LncRNAs regulate neural development by binding to the proximal regions of the protein-coding genes expressions. Most importantly, evolutionarily conserved, nuclearly localized lncRNA Sox1 overlapping transcript (Sox1ot) and Sox2 overlapping transcript (Sox2ot) were identified in the developing brain. Overlapping transcripts Sox1ot and Sox2ot imbricate with Sox1 and Sox2 protein coding transcripts, respectively [79, 80]. Protein coding Sox1 and Sox2 transcripts function as pluripotent transcription factors to maintain the stemness of NPCs and NSCs [81]. Sox1ot and Sox2ot are highly expressed during neural development and are directly proportional to the Sox1 and Sox2 transcription factors abundance respectively [55, 82]. Mechanistically, overlapping transcript Sox2ot interacts with the transcriptional regulator YY1 and binds to CpG island which is present in the proximity of Sox2 locus and subsequently suppresses the Sox2 expression to restrain stemness of NSCs and NPCs [55]. LncRNA rhabdomyosarcoma 2-associated transcript (RMST) binds with promoter regions of Sox2 and regulates the downstream target genes and is critical for neurogenesis [56]. Another, lncRNA Kdm2b is uniquely transcribed from the bidirectional promoter along with Kdm2b. Epigenetically, lncRNA Kdm2b binds in cis manner with hnRNPAB and manages Kdm2b’s transcription. Although lncRNA Kdm2b expression is transient and occurs only during early neuronal differentiation of cortical projection neurons [57]. LncRNA Paupar, regulates the function of transcriptional/epigenetic regulatory factors and modulates neuroblastoma cell growth. Most importantly, it directly binds to KAP1, which induces H3K9me3 methylation and modulates the expression of downstream target genes important for neuronal proliferation and differentiation. LncRNA Paupar epigenetically modulates (by binding H3K9me3 in cis manner) and controls Pax6 mediated neural differentiation and olfactory bulb neurogenesis [58, 59].

11. lncRNA as competing endogenous RNA (ceRNA) against miRNA

LncRNAs participate in neural development via acting as ceRNA of miRNA and indirectly regulate transcript expression in the cytoplasm [61, 83].

LncRNA Gm21284 promotes differentiation of NSCs to hippocampal cholinergic neurons by binding to miR-30e-3p, miR-431 and miR-147 and on silencing miRNAs impedes NSCs proliferation and enhances NSCs differentiation [60]. LncRNA1604 act as sponge to miR-200c and regulates important transcription factor zinc finger E-box binding homeobox1/2 (ZEB1/2) axis which promotes neural differentiation and on silencing miR-200c repress neural differentiation [61]. LncRNA isoforms also play an important role in neurogenesis. For instance, lncRNA RiK has two variants, Rik-201 and Rik-203. Both these variants get activated by CCAAT/enhancer-binding protein β (C/EBPβ) which are induced during neurogenesis. Mechanistically, Rik-201 triggers C/EBPβ, miR-96/Sox6 axis and [62] and lncRNA Rik-203 induces miR-467a-3p/Sox6, miR-101-3a/Glycogen Synthase Kinase-3β (GSK-3β) [62, 63] to promote neural differentiation. LncRNA MEG3 is also engaged in the process of neuron differentiation. Although it acts as a negative regulator of miR-128-3p it induces the cAMP/response element-binding protein (CREB) pathway [64].

12. LncRNAs as key signalling pathway modulators

LncRNAs moreover can contribute to neural differentiation through NSCs and may function as a key member of the signaling pathway. Neurite outgrowth is an essential act in the early neuronal-differentiation and self-renewal. The lncRNA Metastasis-associated lung adenocarcinoma transcript1 (Malat1) is a requisite for neurite growth in in vitro differentiation of neuroblastoma-derived Neuro-2a (N2a) cell as a model. Knockdown of Malat1 impeded neurite outgrowth and enhanced cell death in N2a cells. This owes to suppression of Mitogen-Activated Protein Kinase (MAPK) and activation of Peroxisome proliferator-activated receptor (PPAR) and p53 signalling pathways [65].

13. LncRNAs mediated repression of neuronal differentiation

As described above, most of the highly expressed lncRNAs promote neuronal differentiation, however, some other neuronal lncRNAs were revealed which blocks neuronal differentiation and plays an important role in brain development.

Nuclear localized lncRNA Pnky, was determined to be involved in neuronal development via inhibiting neuronal differentiation. Specifically, Pnky is expressed selectively in neural tissues that are enriched in SVZ-NSCs which are suppressed into mature neurons. Pnky binds to the pre-mRNA splicing regulator RNA-binding protein (RBP) polypyrimidine tract-binding protein (PTBP1). On silencing either Pnky or PTBP1 alters splicing signature of expressed mRNAs in the cell and subsequently induces neurogenesis in SVZ-NSCs. Hence, inverse correlation has been observed between expression of Pnky-PTBP1 complex and neurogenesis [30, 66]. Another lncRNA lncR492 acts as inhibitor of neuroectodermal differentiation via interacting with mRNA binding protein HuR and activating the Wnt signaling pathway [67]. Zhang et al. reported dose-dependent over expression of lncRNA brain derived neurotrophic factor antisense (BDNF-AS), inhibits neural growth in ketamine-treated mouse embryonic NSC-derived neurons. SiRNA mediated silencing of BDNF transcript expression improved neural apoptosis; inhibited neurite growth in NSC-derived neurons through stimulating potassium uptake system protein (TrkB) signaling pathway [68].

14. Role of lncRNAs in regulation of gliogenesis

Recent reports suggest that radial glial (RG) cells are considered for glial lineage along with a subpopulation of astrocytes. RG as matter of fact act as the NSCs that serve as progenitors for many differentiated neurons and glial cells during development and in the postnatal brain give rise to adult SVZ-NSCs that continue to produce neurons throughout adult life [84]. Importantly, at the inception of cortical development, NSCs or NPCs consecutively give rise to deep layer neurons trailed by surficial layer neurons; at later phase of cortical development, NSCs annihilate neurogenesis and move towards gliogenesis to attain gliogenic capability [85, 86]. Temporal NSCs transition from neurogenesis to gliogenesis is a prerequisite for proper cortical development [86, 87].

Several lncRNAs are considered as key regulators during neuronal-glial fate specification and oligodendrocyte lineage maturation. Time dependent overexpression of human urothelial carcinoma associated 1 (UCA1) was able to decide the direction of NSCs differentiation. Knockdown of UCA1 suggested suppression of NSCs proliferation and differentiation with decreased expression of nestin and the enhanced formation of the neurosphere. Further the silencing of UCA1 repressed NSCs differentiation into astrocytes rather NSCs were directed to differentiate as neurons due to the overexpression of miR-1 expression and decreased expression of its target gene-Hes1. Hence, UCA1 regulated the NSCs proliferation and differentiation through regulating Hes1 expression [54].

Dong et al. screened 5000 lncRNAs and identified lncRNAs that are modulated during oligodendrocyte precursor cell (OPC) differentiation from NSCs and play an essential role in oligodendrogenesis. Lnc-OPC was overexpressed in OPCs and is found to be highly conserved among placental mammals and predicts its role in brain development. Mechanistically, lnc-OPC binds to upstream regulatory elements of OLIG2 and is directly proportional to the OLIG2 expression. Hence, overexpression of lnc-OPC enhances OPCs differentiation and oligodendrogenesis [69].

LncRNA has also emerged as an important regulator in oligodendrocyte mediated myelination and plays a crucial role in development and function of CNS [88, 89]. This was supported by dynamic co-expression signature of lncRNAs with protein coding genes at different stages of oligodendrocyte growth and myelination. Most importantly, highly conserved chromatin-associated lncRNA-lncOL1 has been identified during oligodendrocyte growth and myelination. Genetic knockdown of lncOL1 causes aberrations in myelination and remyelination processes after injury, while gain of function induces early oligodendrocyte differentiation i.e., maturation in neural development. Mechanically, lncOL1 forms a complex with a by binding to the promoter region of a member of polycomb repressive complex 2 (Suz12), involved in oligodendrocyte maturation [44]. Another lnc158 upregulates in NSCs and stimulates downstream various oligodendrocyte-related genes expressions including DNA binding transcription factor-nuclear factor-IB (NFIB) that regulates oligodendrocyte lineage differentiation [71]. Additionally, immature OL-specific lncRNA-Pcdh17 is a specific marker for newly born immature OLs and has been identified both in developing and adult forebrain of mice [72]. Interestingly, lncRNA oligodendrocyte maturation-associated long intervening non-coding RNA (OLMALINC) and its antisense counterpart, OLMALINCAS, both are equivalently and abundantly expressed in the white matter of human frontal cortex as opposed to grey matter and peripheral tissues and basically take part in modulation of human oligodendrocyte maturation related genes. OLMALINCAS, maps to the first exon of the major isoform of OLMALINC [73].

15. The role of lncRNAs in synaptogenesis

Synaptic stability in the developing and adult nervous system results due to the late phase long-term potentiation i.e., a continuous strengthening of synapses for long lasting increase in signal transmission between two neurons. The role of lncRNAs in modulating synaptic stability is ambiguous. Wang et al. [74], reported that cerebellum of the brain shows increased expression of lncRNA, Synage, to regulate synaptic stability. The lncRNA mediated synaptic stability is either lncRNA acting as a sponge or as a scaffold. As a sponge lncRNA Synage binds to miR-325-3p and alters the expression of downstream cerebellar synapse organizer identified in mouse, rhesus macaque, and human. Additionally, lncRNA Synage serves as a scaffold for rearranging the positioning of the LRP1-HSP90AA1-PSD-95 complex in Parallel fibre (PF)-Purkinje cell (PC) synapses. Knockdown of synage collapses cerebellar phenotype and leads to cerebellar degeneration, death of neurons, decline in synapse density which relates to synaptic pruning, decreased synaptic growth and synaptic plasticity during cerebellar development. Hence, the lncRNA Synage plays a major role in regulating synaptic stability and plays a crucial role during cerebellar development. GM12371 (nuclear enriched [75]) and Gm2694 (cerebellar cortex enriched [76]) (alias AK082312) also acts as a transcriptional regulator of synapse function.

16. Conclusion

Neural development related to NSCs/NPCs which constructs embryonic tissue architecture of CNS as well as exist as remnant in subventricular zone (SVZ) nearby the lateral wall of the lateral ventricles and the subgranular zone (SGZ) of hippocampus dentate gyrus (DG) of the brain is considered as a complex phenomenon. Advanced large-scale genome-wide RNA sequencing has been performed in various neuronal cells over the course of neural development to understand NSC self-renewal, neurogenesis, gliogenesis and synaptogenesis. This review has described in detail the functional roles of lncRNAs as ceRNA, by regulating proximal protein-coding genes expressions, and epigenetic modulations in regulation of NSCs/NPCs self-renewal, proliferation and differentiation into neuron or glial cells and synaptogenesis. This suggests that lncRNAs might be employed as potential selection biomarkers for identifying or screening suitable NPCs/NPCs. Importantly, a spatiotemporal expression of lncRNA as atlas of embryonic neurogenesis in Drosophila revealed a high- confidence set of 13 lncRNAs will open a new era of lncRNA based NSCs mediated neurogenesis and may help us to better understand the neuronal physiology. However, most of their function remains to be explored, more novel lncRNAs and their molecular mechanisms remain to be found and probed in-depth yet.

Acknowledgments

A special thanks to my Dr. Sanjay Kumar Singh for inspiring me to write this article and for financial support.

Abbreviations

Aldh1l1	aldehyde dehydrogenase 1 family member L1
AR	androgen receptor
BDNF	brain-derived neurotrophic factor
C/EBPβ	CCAAT/enhancer-binding protein β
ceRNA	ZEB1/2zinc finger E-box binding homeobox1/2
CNS	central nervous system
CO	cerebral cortex organoid
CP	cortical plate
CR	Cajal-Retzius
CREB	cAMP/response element-binding protein
CTIP2	COUP-TF-interacting protein 2
DCX	doublecortin
DG	dentate gyrus
Dlx2	Distal-Less Homeobox 2
EGL	external granule layer
ERK	extracellular signal regulated kinase
ESCs	embryonic stem cells
GSK-3β	glycogen synthase kinase-3β
IGL	internal granule layer
IPCs	intermediate progenitors
IZ	intermediate zone
Kdm2b	lysine demethylase 2B
LncRNAs	long non-coding RNAs
LV	lateral ventricle
GFAP	glial fibrillar protein
Malat1	metastasis-associated lung adenocarcinoma transcript1
MAPK	mitogen-activated protein kinase
MEG3	maternally expressed gene 3
MSNP1AS	Moesin pseudogene 1 antisense
N2a	Neuro-2a
NE	neuroepithelium
NEAT1	nuclear paraspeckle assembly transcript 11
NeuN	neuronal nuclei
NFIB	nuclear factor-IB
NG2	polydendrocytes
Notch	Notch receptor
NPCs	neural precursor/progenitor cells
NSCs	neural stem cells
OB	olfactory bulb
OCT3/4	POU class 5 homeobox 1
Olig2	oligodendrocyte transcription factor
OPC	oligodendrocyte precursor cells
OLMALINC	oligodendrocyte maturation-associated long intervening non-coding RNA
Paupar	PAX6 Upstream Antisense RNA
PAX6	Paired Box 6
PC	Purkinje cell
PDGF	platelet-derived growth factor
Pnky	long intergenic non-protein coding RNA PNKY
PF	parallel fiber
PPAR	peroxisome proliferator-activated receptor
Prox1	Prospero Homeobox 1
PSC	pluripotent stem cell
PTBP1	RNA-binding protein (RBP)-polypyrimidine tract-binding protein
RGLs	radial glia-like cells
RBP	RNA-binding protein
RG	radial glial
RMS	rostral migratory stream
RMST	rhabdomyosarcoma 2-associated transcript
RPS10P2-AS1	ribosomal protein S10 pseudogene 2 anti-sense 1
S100β	S100 calcium-binding protein B
SGZ	sub-granular zone
SHH	sonic hedgehog
Sox10	SRY-related HMG-box 10
Sox1ot	Sox1 overlapping transcript
Sox2ot	Sox2 overlapping transcript
Suz12	polycomb repressive complex 2
SVZ	subventricular zone
TALNEC2	tumor associated lncRNA expressed in chromosome 2
TBR1	T-box brain transcription factor 1
TBR2	T-box brain transcription factor 2
TGF-β	transforming growth factor-β
TREX	transiently expression of lncRNAs
Trincr1	TRIM71 interacting long noncoding RNA 1
TUNA	Tcl1 upstream neuron-associated lincRNA
UCA1	urothelial carcinoma associated 1
Wdr5	WD repeat domain 5
ZEB1/2	Zincfinger E-box binding homeobox ½