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How long does Lentivirus take to express in vivo mouse neurons?

How long does Lentivirus take to express in vivo mouse neurons?


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Does anyone know how long it takes for a standard Lentivirus vector to express its genes (under a strong promoter such as CAG, CB7, etc.), after injection into the brain of a mouse?

By hearsay I think it's 2 weeks for full expression, is that true?


This depends a lot on what you are expressing (such as how stable it is), and where you need it (soma vs dendrites vs axons, and whether it is trafficked actively in the cell).

For optogenetic constructs, our lab has typically seen near-peak expression around 3 weeks, though expression may be suitable earlier, and toxic later.


How long does Lentivirus take to express in vivo mouse neurons? - Biology

Lentiviral vectors can deliver and express genes in a wide variety of dividing and nondividing cells. These include terminally differentiated neurons, myotubes, hepatocytes, and hematopoietic stem cells. We now describe the generation of lentiviral vectors in which the expression of the transgene can be regulated. We have developed an inducible lentiviral vector system that contains the entire tetracycline (Tet)-regulated system developed by H. Bujard and colleagues. The novel vector expresses the GFP reporter gene and the tetracycline transactivator under the control of the tetracycline-inducible promoter and the human CMV promoter, respectively. In vitro transduction of human 293 cells resulted in a very low basal expression of GFP in the presence of the effector substance doxycyline. Withdrawal of doxycyline induced a more than 500-fold increase in transgene expression. Switching transgene expression "off and on" did not change either the kinetics or the magnitude of induction. Maximal suppression of GFP mRNA transcription was achieved within 24 h of addition of the drug however, due to the slow turnover rate of GFP, green fluorescent cells could be detected up to 10 days following doxycyline treatment. Following transduction of rat brain with recombinant lentiviruses, doxycyline-regulated GFP expression could be observed in terminally differentiated neurons. Specifically, by adding or withdrawing doxycyline from the rats' drinking water, induction and suppression of GFP expression could be regulated in vivo. These studies show that an inducible lentiviral vector can deliver and regulate transgene expression in vivo. We believe that regulated gene expression is an essential tool for successful gene therapy approaches.


2nd Generation

The graphic to the right shows how the lentiviral genome is edited down and distributed across the three plasmids comprising the 2nd-generation lentiviral system. This system contains a single packaging plasmid encoding the Gag, Pol, Rev, and Tat genes. The transfer plasmid contains the viral LTRs and psi packaging signal (not pictured). Unless an internal promoter is provided, gene expression is driven by the 5'LTR, which is a weak promoter and requires the presence of Tat to activate expression. The envelope protein Env (usually VSV‐G due to its wide infectivity) is encoded on a third, separate, envelope plasmid. All 2nd generation lentiviral transfer plasmids must be used with a 2nd generation packaging system because transgene expression from the LTR is Tat-dependent.

Second Generation Lentiviral Plasmids

Pseudotype-dependent lentiviral transduction of astrocytes or neurons in the rat substantia nigra

Gene transfer to the central nervous system provides powerful methodology for the study of gene function and gene–environment interactions in vivo, in addition to a vehicle for the delivery of therapeutic transgenes for gene therapy. The aim of the present study was to determine patterns of tropism exhibited by pseudotyped lentiviral vectors in the rat substantia nigra, in order to evaluate their utility for gene transfer in experimental models of Parkinson's disease. Isogenic lentiviral vector particles encoding a GFP reporter were pseudotyped with envelope glycoproteins derived from vesicular stomatitis virus (VSV), Mokola virus (MV), lymphocytic choriomeningitis virus (LCMV), or Moloney murine leukemia virus (MuLV). Adult male Lewis rats received unilateral stereotactic infusions of vector into the substantia nigra three weeks later, patterns of viral transduction were determined by immunohistological detection of GFP. Different pseudotypes gave rise to transgene expression in restricted and distinct cellular populations. VSV and MV pseudotypes transduced midbrain neurons, including a subset of nigral dopaminergic neurons. In contrast, LCMV- and MuLV-pseudotyped lentivirus produced transgene expression exclusively in astrocytes the restricted transduction of astroglial cells was not explained by the cellular distribution of receptors previously shown to mediate entry of LCMV or MuLV. These data suggest that pseudotyped lentiviral vectors will be useful for experimental gene transfer to the rat substantia nigra. In particular, the availability of neuronal and astrocytic-targeting vectors will allow dissociation of cell autonomous and cell non-autonomous functions of key gene products in vivo.

Research Highlights

►Lentiviral tropism in the rat substantia nigra is pseudotype-dependent. ►VSV- and Mokola-pseudotyped lentiviruses selectively transduce midbrain neurons, including nigral dopamine neurons. ►Neither pseudotype induces signs of toxicity in the nigrostriatal system. ►LCMV- and MuLV-pseudotyped lentiviruses selectively transduce midbrain astrocytes rather than neurons. ►Astrocytic tropism of LCMV- and MuLV-pseudotyped vectors is not explained by the cellular distribution of receptors known to mediate entry of native LCMV or MuLV.


12.5. CONCLUSIONS

In this chapter, we have briefly described the use of a viral-based gene expression system to acutely express heterologous proteins in the rat brain in vivo. We believe that this technique offers several advantages over other approaches that are used to express recombinant proteins in vivo. In particular, it allows evaluation of the effect of acute expression of any protein of interest in a temporally and spatially restricted manner while minimizing the possibility of time-dependent compensations in response to the molecular manipulation. It also permits direct comparison of molecularly-manipulated and neighboring control neurons within the same tissue under close-to-ideal physiological conditions. However, one drawback of the use of Sindbis viruses is that they often result in high over-expression of the recombinant protein. In theory, this could affect the normal functioning of the neuron as well as result in the recombinant protein having effects that the endogenous protein does not. Thus, we encourage the reader to keep up-to-date with the latest versions of the Sindbis viruses that show the least cytotoxicity and to consider the use of other viral-expression systems such as lentiviruses, which permit lower-level and longer-term expression of recombinant proteins. Lentiviruses can be particularly advantageous when using it to express RNAi to knockdown the expression of endogenous proteins.


Lentivirus vector selection guide

We offer a variety of highly optimized lentiviral vectors for many applications. These lentiviral vectors are key players in all of our Lenti-X gene delivery systems, which can be used with almost any mammalian cell type, including dividing and nondividing cells, primary cell cultures, stem cells, and neurons. All of our pLVX lentiviral vectors contain HIV-1 LTRs and the lentiviral packaging signal (&Psi), and we've included specific elements to improve transgene expression, virus titer, and overall lentiviral vector function:

  • WPRE: A woodchuck hepatitis virus posttranscriptional regulatory element prevents poly(A) site readthrough, promotes RNA processing and maturation, and increases nuclear export of RNA. In genomic transcripts, it enhances vector packaging and increases titer. In transduced target cells, the WPRE boosts transgene expression by facilitating mRNA transcript maturation.
  • cPPT/CTS: A central polypurine tract/central termination sequence creates a "DNA flap" that increases nuclear importation of the viral genome during target-cell infection. The cPPT/CTS element improves vector integration and transduction efficiency.
  • RRE: A Rev response element increases titers by promoting the nuclear export of unspliced viral genomic RNA.

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Use these lentiviral vectors with any of our highly advanced, safe, and easy-to-use 4 th -generation Lenti-X packaging systems, which produce high titers of VSV-G- or ecotropic-pseudotyped lentivirus.

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Express your gene of interest from a strong CMV promoter, and select for lentiviral integration using antibiotic selection.

For cell types where the CMV promoter gets silenced, such as hematopoietic or stem cells.

Co-express your gene of interest and an antibiotic or fluorescent selectable marker from the same transcript using these IRES-containing lentiviral vector systems.

Obtain tightly controlled, inducible expression of your gene of interest with a single vector using Tet-One systems.

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Deciphering the function of factors involved in spinal cord neuroinflammation and tissue plasticity associated with neuropathic pain

Pathological pain is characterized by extensive modification of the systems involved in pain signal transmission and modulation at the spinal level (primary sensory neurons and the spinal cord) and probably in the brain. Chronic pain, particularly of neuropathic origin, may also lead to tissue remodeling (plasticity). This may include, for instance, loss of spinal interneurons, abnormal rearrangement of central afferents of primary sensory neurons and glial cell activation and proliferation. These long-lasting modifications are mediated by, or associated with, changes in the production of key molecules involved in nociceptive processing. Gene-based techniques allow local or even cell-type-specific interventions to be used to correct the abnormal production of some of these proteins, modulate the activity of signal transduction pathways or overproduce various therapeutic secreted proteins. In fact, with these approaches, it may be possible to not ‘only’ relieve established ongoing pain but to reverse the pathological situation underlying chronic pain.

The importance of changes in glial cell status (so-called activation) in the spinal cord, 1, 2, 3, 4 dorsal root ganglia (DRG) 5 and even brain structures 6, 7 in generating and also probably maintaining neuropathic pain is now well established. Indeed, activation of spinal cord glia has been associated with almost all animal models of neuropathic pain (see L Watkins section in this volume). Activated glia (microglia and astrocytes) produces and releases several molecules (gliotransmitters) that may act directly on sensory neurons (modifying the excitability of both first- and second-order neurons) and/or glial cells themselves (maintaining their high level of activity). Although alteration of glial cell function clearly participates in the development of long-term neuroinflammation in the spinal cord, thus having an important function in pathological pain, the precise mechanisms involved are still a matter of debate. Pharmacological studies have implicated numerous receptors, transduction pathways and secreted mediators in central sensitization in the spinal cord following peripheral nerve injury. However, tools need to be developed to determine the relative input of glia and neurons. Indeed, both neurons and glia express several receptors potentially involved in glia activation (for example, receptors for certain cytokines, chemokines, glutamate, ATP, substance P) and in both cell types several signal transduction pathways are also activated following peripheral nerve injury (for example, p38 MAP kinase, 8 JNK 9 and ERK 10 ). The actual source of pro-inflammatory and algogenic mediators present in the spinal cord after peripheral nerve lesion is difficult to determine, given that most of them (glutamate, cytokine IL-6, chemokine CCL2, ATP, nitric oxide, prostaglandins and so on) may be synthesized in and released from both neurons and glial cells. Thus, separate in vivo studies of glia or neurons, using different models of chronic pain and distinguishing between peripheral (especially those involving primary sensory neurons) and central mechanisms, will be needed to determine the precise nature and chronological order of the events leading to the development and maintenance of pain. Classical pharmacological approaches are not sufficient to fulfill such criteria. Indeed, the selectivity of some molecules is only relative, local delivery is problematic due to the substantial diffusion of agents, intrathecal delivery of various compounds frequently leads to their massive accumulation in pia mater and in DRG, 9, 11, 12 prolonged delivery of drugs is only possible with minipump systems, leading to a broad and diffuse action of the drug, and cell-selective treatment is almost impossible.

Molecular mechanisms involved in the activation of spinal cord glia and/or the induction of glia-derived deleterious products undoubtedly represent therapeutic targets of potential interest. It may be possible to use local and cell-type-selective interventions to modulate some of these cellular mechanisms in the spinal cord glia and to determine the importance of these mechanisms in the modification of glial cell function, production of algogenic mediators and, ultimately, in chronic pain. This approach would require a direct intraparenchymal injection of vectors rather than intrathecal administration, which tends to the predominant transduction of meningeal cells or DRG sensory neurons and only very modest transduction of spinal cord tissue. 13, 14, 15 Additionally, central nervous system (CNS) intraparenchymal injection of vector limits the systemic immune response, thus inducing only a negligible inflammatory response. 16


Acknowledgements

We would like to thank Dr. Hiromu Yawo, Michisuke Yuzaki and Atsushi Miyawaki for providing materials, the FCK-Halo-GFP construct and the pCS2-Venus plasmid, respectively. This work was supported by grants from JSPS KAKENHI (16H04675), the JSPS Core-to-Core Program, A. Advanced Research Networks, and a research grant from The Takeda Science Foundation to S.T., and a grant from JSPS KAKENHI (16K18397) to Y.E. Finally, we would like to thank Editage (www.editage.jp) for English language editing.


Results

Inducing neurogenesis in the adult spinal cord

We used a lentiviral gene delivery system to target both proliferating and quiescent cells in the adult mouse spinal cord. Gene expression was regulated by the human glial fibrillary acidic protein (hGFAP) promoter, which is active primarily in astrocytes 30 . To further examine the cell types targeted by this lentiviral system in the adult spinal cord, 1.5 μl of green fluorescent protein (GFP)-expressing virus (hGFAP-GFP) was injected at two positions 3–5 mm apart at the thoracic level 8 (T8). One week post viral injection (wpi), immunohistochemical analyses of longitudinal sections around the injected regions showed that GFP + cells were detectable in a broad area, especially in the white matter, reaching a distance of

3.0 mm from the injection site both rostrally and caudally (Supplementary Fig. 1a). While a few GFP + cells were stained positive for markers of neurons (NeuN, <0.82%), oligodendrocyte precursors and pericytes (OLIG2, 4.94±3.10% and NG2, 4.36±2.83% mean±s.d., n=3), the vast majority expressed the astrocyte-specific marker GFAP (95.09±4.15%, mean±s.d., n=3) (Supplementary Fig. 1b–e,i). Markers for mature oligodendrocytes (MBP and PLP) or microglia (IBA1) were not detected in GFP + cells (Supplementary Fig. 1f–i). These results indicate that spinal astrocytes are the major cell type targeted by lentivirus under the regulation of hGFAP promoter.

On the basis of their roles in NSCs and/or neurogenesis, 12 genes (SOX2, PAX6, NKX6.1, NGN2, ASCL1, OLIG2, SOX11, Tlx, OCT4, c-MYC, KLF4 and PTF1a) were chosen as candidates. Lentivirus expressing these candidates under the hGFAP promoter was individually injected into the T8 region of the adult spinal cord and analysed for their ability to induce adult neurogenesis (Fig. 1a). Neurogenesis was initially examined by staining for the expression of doublecortin (DCX), a microtubule-associated protein that is broadly expressed in neuroblasts and immature neurons during development and in neurogenic regions of the adult brain 31,32 . DCX expression is mainly associated with adult neurogenesis but not with reactive gliosis or regenerative axonal growth 33 . Consistent with these results, DCX was not detected in either intact spinal cords or those with hemisection-induced injuries (Supplementary Fig. 2). In sharp contrast, DCX + cells were identified in spinal cords injected with virus expressing SOX2 but none of the other 11 candidate genes at 4 wpi. This was further confirmed with a virus expressing GFP-T2A-SOX2 so that virus-transduced cells could be identified by the coexpression of GFP (Fig. 1b–d).

(a) Experimental scheme. (b) DCX + cells are detected in animals injected with lentivirus expressing SOX2 but not control GFP at 4 or 5 wpi. Nuclei were counterstained with Hoechst 33342 (Hst) (c,d) Quantification of SOX2-induced DCX + cells around the virus-injected regions in adult spinal cords (mean+s.d. n=3 mice per group n.d., not detected). (e) Confocal images of a representative DCX + cell (indicated by arrows). SOX2-induced DCX + cells are traced by the coexpressed GFP, indicating an origin of virus-infected cells. They are also co-labelled by TUBB3 and have bipolar or multipolar processes. Asterisks and arrowheads indicate cell bodies and neuronal processes, respectively. (f) Representative images of DCX + cells induced in the spinal cord of aged mice (>12 months). Scale bars, 50 μm (b,f) and 20 μm (e).

SOX2-induced DCX + cells were mainly identified surrounding the virus-injected region and showed typical immature neuronal morphology with bipolar or multipolar processes (Fig. 1b,e). They coexpressed betaIII-tubulin (TUBB3, also known as TUJ1), a pan-neuronal marker, and were labelled by GFP, showing an origin of virus-transduced cells (Fig. 1e). The induction efficiency of DCX + cells was estimated at 6–8% of GFP + cells surrounding the core injection sites at 4 or 5 wpi (Fig. 1d). Interestingly, ectopic SOX2 also resulted in the production of DCX + cells in aged mice (>12 month, Fig. 1f). Together, these data suggest that neurogenesis can be induced by a single transcription factor, SOX2, in the adult spinal cord, similar to observations made in the adult striatum 29 .

Inducing neurogenesis in spinal cords with severe injuries

Severe traumatic injury to the adult spinal cord causes massive cell death, inflammation and gliosis 1,25,34 , which result in a pathological microenvironment drastically different from that of the needle injection-induced stab wound injury. To examine whether neurogenesis could also be induced under this clinically-relevant pathological condition, we injected lentivirus into the parenchyma of severely injured spinal cord immediately after hemisection at the T8 level (Fig. 2a). The two injection sites were 1.5 mm away from the injury core on each side (Supplementary Fig. 3a). Histological analyses were performed on spinal cord sections spanning the lesion site. Similar to what was observed in the intact spinal cord, the control virus hGFAP-GFP could efficiently transduce cells surrounding the injection sites with a majority expressing the astrocyte marker GFAP (95.21±3.95%, mean±s.d., n=3 Supplementary Fig. 3b,i). Only a small percentage of GFP + cells expressed markers for neurons, oligodendrocyte precursors or pericytes (NeuN + , <0.91% OLIG2, 4.73±2.94% NG2, 3.73±2.74% mean±s.d., n=3) (Supplementary Fig. 3c–e,i). None expressed the markers for mature oligodendrocytes MBP and PLP or the microglia marker IBA1 (Supplementary Fig. 3f–i). These results are very similar to those in spinal cords without the hemisection (Supplementary Fig. 1), suggesting that severe injury does not change the cell types targeted by lentivirus.

(a) Experimental scheme. Lentivirus was injected into the spinal cord immediately after hemisection at the T8 level. (b) Quantification of SOX2-induced DCX + cells around the virus-injected regions in injured spinal cords (mean+s.d. n=4 mice per group n.d., not detected). (c) Percentage of GFP + cells expressing DCX around the virus-injected regions in injured spinal cords (mean+s.d. n=4 mice per group n.d., not detected). (d) Representative images of DCX + cells induced by virus expressing SOX2 but not the GFP control at 4, 6 or 8 wpi. Scale bar, 40 μm.

No DCX + cells were detected in the injured spinal cords injected with the control virus hGFAP-GFP at 4, 6 or 8 wpi (Fig. 2b–d). In stark contrast, these cells were specifically induced by the injection of hGFAP-GFP-T2A-SOX2 virus (Fig. 2b–d). All the induced DCX + cells also expressed GFP indicating an origin from virus-transduced cells (Fig. 2d). An estimation of 3–6% of GFP + cells surrounding the core viral injection sites were reprogrammed by SOX2 to become DCX + cells between 4–8 wpi (Fig. 2c). These DCX + cells also stained positive for the neuronal marker TUBB3 (Fig. 2d). Together, these data indicate that neurogenesis can be induced by SOX2 in an injured environment of the adult spinal cord.

SOX2-induced neurogenesis originates from spinal astrocytes

The cellular source for SOX2-induced DCX + cells was determined by genetic lineage tracing. Gene expression under the hGFAP promoter was not detected in mature oligodendrocytes or microglia (Supplementary Figs 1,3), thus excluding them as a possible origin for SOX2-induced DCX + cells. Although less than 5% of NG2 + cells were targeted by the hGFAP promoter (Supplementary Figs 1,3), these cells were specifically examined as they are cycling precursors for oligodendrocytes, exhibit plasticity after injury in the adult central nervous system 35 and are amenable to fate reprogramming in culture 15 . NG2 + cells and their derivatives were traced using Ng2-Cre BAC transgenic mice 36 and the reporter Rosa-YFP (Fig. 3a). A respective 71 and 64% of YFP + cells expressed NG2 and OLIG2 in the adult spinal cord (Supplementary Fig. 4a,b,d). Markers for astrocytes (GFAP), microglia (IBA1) or neurons (TUBB3) were not detected in YFP-traced cells (Supplementary Fig. 4c,d). Transgenic mice were then injected with hGFAP-SOX2 lentivirus and examined at 5 wpi. Immunohistochemistry showed that DCX + cells were induced around the virus-injected regions but none co-labelled with YFP (Fig. 3b). This result indicates that DCX + cells induced by SOX2 under the hGFAP promoter do not originate from NG2 + cells in the adult spinal cord.

(a,b) SOX2-induced DCX + cells do not come from NG2 + cells. Ng2-CreRosa-YFP mice (a) were injected with SOX2-expressing or empty lentivirus and were analysed at 5 wpi. Confocal images are shown in panel (b). (cg) SOX2-induced new neurons originate from astrocytes. (c) mGfap-CreRosa-tdT mice were injected with virus expressing either GFP-T2A-SOX2 or GFP (as a control) and were analysed at 5 wpi. (d) Representative confocal images showing that SOX2-induced DCX + cells originate from lentivirus-infected astrocytes (indicated by GFP + tdT + ). (e) Higher magnification views of a tdT-labelled DCX + cell. Asterisks and arrowheads indicate cell bodies and processes, respectively. (f,g) SOX2-induced TUBB3 + neurons are derived from virus-infected astrocytes (indicated by GFP + tdT + ). Traced neurons were quantified in virus-injected regions (mean+s.d. n=5 mice per group n.d., not detected). Scale bar, 20 μm (b,df).

In contrast, astrocytes are the most likely cellular origin for the induced neurons since they are the predominant cell type targeted by lentivirus under the hGFAP promoter (Supplementary Figs 1,3). This hypothesis was examined by genetic lineage tracing using the transgenic mGfap-Cre line 77.6, which was reported to exclusively trace astrocytes in the forebrain 23 . We crossed this line to Rosa-tdTomato (tdT) reporter 37 and examined the identity of the labelled cells in the adult spinal cord (Fig. 3c, Supplementary Fig. 5). On the basis of GFAP expression, 71.32±6.75% of astrocytes were labelled by the reporter tdT. Among tdT + cells, the vast majority expressed the astrocyte markers GFAP (97.36±3.24%, mean±s.d., n=3) and glutamine synthetase (GS, 95.14±3.86%, mean±s.d., n=3), while fewer than 5% were positive for NG2 or OLIG2 (Supplementary Fig. 5a–d,f). Markers for microglia (IBA1 + ) or neurons (NeuN + , MAP2 + and TUBB3 + ) were not expressed in traced cells (Supplementary Fig. 5e,f). Adult mGfap-CreRosa-tdT mice were then injected with lentivirus expressing either GFP (as a control) or GFP-T2A-SOX2 and examined at 5 wpi. Immunohistochemistry showed that ectopic SOX2 induced DCX expression in tdT + cells (Fig. 3d). Importantly, a fraction of the traced cells were also labelled by the neuronal marker TUBB3 (

5%) in the core spinal regions injected with SOX2 virus (Fig. 3f,g). In stark contrast, these neuronal markers were not detectable in tdT-traced cells in spinal cords injected with the control virus (Fig. 3g), although GFP expression showed a clear infection of these cells (Supplementary Fig. 6). These results indicate that SOX2-induced new neurons come from resident astrocytes.

Cell transplantation assays were performed to further confirm these results. Spinal astrocytes were isolated from neonatal mGfap-CreRosa-tdT mice, cultured for 7–10 days in serum-containing medium, and subsequently passaged once. tdT + astrocytes were then purified by fluorescence-activated cell sorting and infected with either hGFAP-GFP or hGFAP-GFP-T2A-SOX2 lentivirus (Fig. 4a). The astrocytic identity and expression of ectopic GFP and SOX2 were confirmed by immunocytochemistry (Fig. 4b,c). Three days post viral infection (dpi), these cells were transplanted into the spinal cords of adult immunodeficient NOD scid gamma (NSG) mice. When examined at 4 wpi, none of the control GFP virus-infected tdT + cells stained positive for the neuronal marker TUBB3 (Fig. 4d). In contrast, TUBB3 expression was clearly detectable in tdT + cells that were infected with the hGFAP-GFP-T2A-SOX2 virus before transplantation (Fig. 4d). GFP expression in the latter group was also downregulated, reflecting much reduced activities of the hGFAP promoter in converted cells. By 5 wpi, some of the tdT + cells in the SOX2 group started to express MAP2, a marker for mature neurons. These results demonstrate that the ectopic expression of SOX2 can convert transplanted spinal astrocytes to neurons in the adult spinal cord.

(a) Experimental scheme. Spinal astrocytes cultured from mGfap-CreRosa-tdT mice were purified based on tdT expression. Three dpi, cells were infected with lentivirus and transplanted into the spinal cord of NSG mouse. (b) Immunocytochemistry showing the expression of GFAP and Aldolase c (Aldc) markers for astrocytes, in purified tdT + cells. (c) Immunocytochemistry confirming SOX2 expression in spinal astrocytes infected with the hGFAP-GFP-T2A-SOX2 virus. (d) Confocal images showing astrocyte-derived neurons in the spinal cord transplanted with the SOX2 but not control virus-infected astrocytes. Orthogonal views of cells in the boxed regions are shown in the right panels. Astrocyte-derived neurons (tdT + TUBB3 + or tdT + MAP2 + ) are indicated by arrows. Scale bars, 60 μm (b,c) and 30 μm (d).

Cell proliferation during induced neurogenesis

SOX2-induced neurons could be converted from astrocytes by a direct lineage switch without the addition of new cells. We tested this hypothesis by examining cell proliferation during the process of SOX2-induced neurogenesis. Proliferating cells in the injured adult spinal cord were continually labelled by intraperitoneal injection of 5-bromo-2'-deoxyuridine (BrdU) (100 mg kg −1 , twice a day) from 3–18 days post viral injection. When examined at 4 wpi, around 90% of induced DCX + cells were clearly labelled by BrdU indicating that they passed through a proliferative stage (Fig. 5a,c). Interestingly, nearly 17% of DCX + cells also expressed the cell proliferation marker Ki67, indicating that these cells were still in a cycling state (Fig. 5b,c). Using TUBB3 as an additional marker for neurons, we found that

3% of neurons surrounding the core viral injection sites could be labelled by BrdU (Fig. 5d,f). Administration of BrdU during 4–8 wpi also led to a similar number of TUBB3 + cells being labelled (Fig. 5e,f). These data collectively demonstrate that adult neurogenesis induced by the ectopic expression of SOX2 is a continuous process, which passes through a proliferation phase.

(a) Incorporation of BrdU in SOX2-induced DCX + cells. An orthogonal view of BrdU + DCX + cells in the boxed region is shown in the right panel (wk, weeks IHC, immunohistochemistry). (b) Ki67-staining showing that SOX2-induced DCX + cells are proliferative at 5 wpi. An orthogonal view of Ki67 + DCX + cells in the boxed region is shown in the right panel. (c) Quantification of BrdU or Ki67-labelled DCX + cells (mean+s.d. n=3 mice per group). (d,e) Newly generated neurons indicated by BrdU-traced TUBB3 + cells in the adult spinal cord. Orthogonal views of BrdU + TUBB3 + cell are also shown. (f) Quantification of SOX2-induced new neurons (indicated by BrdU + TUBB3 + ) surrounding the virus-injected regions in adult spinal cords (mean+s.d. n=3 mice per group n.d., not detected). Scale bar, 20 μm (a,b,d,e).

SOX2 induces neuroblasts in the adult spinal cord

Ectopic expression of SOX2 alone was able to convert fibroblasts or human cortical astrocytes to proliferative and multipotent NSCs 28,38 , raising the possibility that spinal astrocytes could be similarly reprogrammed by SOX2. Spinal astrocytes were isolated from early postnatal mGfap-CreRosa-tdT mice and infected with lentivirus expressing either GFP-T2A-SOX2 or the control GFP alone (Fig. 6a,b). Three days later, the fetal bovine serum-containing medium for astrocytes was switched to complete synthetic medium for NSCs. Neurospheres, which indicate the presence of self-renewable NSCs, were observed as early as 7 dpi in cultures with ectopic SOX2 but not the control GFP (Fig. 6c,d). These spheres were clearly labelled by tdT and GFP indicating an origin for SOX2 virus-infected astrocytes. Consistent with neural stem-like cells being present in postnatal spinal cords 39 , neurospheres were also detectable in the GFP group by 14 dpi. However, their number was significantly lower than that in the SOX2 group (Fig. 6d). Interestingly, ectopic SOX2 further dramatically enhanced the generation of secondary neurospheres from the primary ones (Fig. 6c,d). These spheres and the single cells dissociated from them were labelled by tdT, GFP and the NSC marker nestin (NES Fig. 6e). When cultured under differentiation conditions for 6 days, neurosphere-derived single cells could become TUBB3 + neurons, GFAP + astrocytes and NG2 + oligodendrocyte precursors (Fig. 6f). All of these cells were traced by the marker tdT indicating an origin from astrocytes. Together, these data show that SOX2 can promote the generation of multipotent NSCs from spinal astrocytes in culture consistent with results from cultured fibroblasts and cortical astrocytes 28,38 .

(a) Experimental scheme. (b) Immunocytochemistry confirming the astrocyte identity of cultured tdT + cells. (c) Representative micrographs of neurospheres from spinal astrocytes infected with the hGFAP-GFP-T2A-SOX2 lentivirus. (d) Frequency of neurosphere-forming cells from spinal astrocytes infected with the indicated lentivirus. Secondary spheres were quantified at 7 days after plating (mean+s.d. n=5 *P<0.01 by Student’s t-test). (e) Nestin (NES) expression in SOX2-induced neurospheres. (f) Multiple lineage differentiation of SOX2-induced secondary neurospheres. Immunocytochemistry was performed 6 days after plating dissociated single cells. Scale bars, 50 μm (b), 200 μm (c), 60 μm (e) and 40 μm (f).

We then examined the SOX2-induced neurogenesis in the adult spinal cord using immunohistochemistry. A time course analysis showed that DCX + cells were not readily detectable until 4 wpi suggesting that the induced neurogenesis was a slow process. SSEA1 (also known as LeX or CD15) and KLF4, markers for multipotent stem cells, were not expressed in virus-infected astrocytes or the induced DCX + cells in the adult spinal cord at 1, 2, 3 or 4 wpi (Fig. 7a). Interestingly, SOX1 and SOX3, members of the SOXB1 subfamily of SOX transcription factors and key players in neural progenitors, were expressed in a subset of SOX2 virus-infected astrocytes, although these two factors were also distributed in some of the non-infected cells (Fig. 7b). The expression of SOX1 and SOX3 persisted into the DCX + stage, consistent with their reported expression in neuroblasts in the subventricular zone of the adult mouse brain 40,41 .

(a) Markers for multipotent stem cells are not detectable in the spinal cord injected with the hGFAP-GFP-T2A-SOX2 virus. The expression in NSCs was served as a control. (bd) The SOXB1 transcription factors are expressed during the reprogramming process. (b) Expression of SOX1 and SOX3 in SOX2 virus-infected cells and induced neuroblasts (indicated by arrows). Cultured NSCs served as controls. (c) Robust expression of SOX2 in the spinal cord injected with the hGFAP-GFP-T2A-SOX2 virus (indicated by arrows). Arrowheads indicate endogenous SOX2 expression. (d) Downregulation of SOX2 in converted neuroblasts (indicated by arrows). Arrowheads indicate endogenous SOX2 expression. Scale bar, 40 μm (ad).

Although endogenous SOX2 is broadly expressed in astrocytes in the adult spinal cord 42 , immunostaining showed that cells infected with the hGFAP-GFP-T2A-SOX2 virus had a significantly higher level of total SOX2 expression when compared with the surrounding non-infected cells (Fig. 7c). In induced DCX + cells, SOX2 expression was downregulated to a level similar to their neighbouring non-DCX + cells at 4 wpi and became almost non-detectable at 6 wpi (Fig. 7d). We hypothesized that such dynamic expression of SOX2 that was delivered by the hGFAP promoter was critical for induced neurogenesis. This hypothesis was tested by controlling SOX2 expression through the constitutively active CMV early enhancer/chicken beta actin (CAG) promoter. Robust SOX2 expression was observed in virus-infected cells however, DCX + cells were not detected at 4 wpi (Supplementary Fig. 7). These results suggest that the initial higher level of SOX2 expression and its subsequent downregulation was indeed necessary for the reprogramming process.

In summary, these results reveal that the ectopic expression of SOX2 reprograms resident astrocytes to proliferative DCX + neuroblasts in the adult spinal cord contrary to in vitro cell culture conditions that enable SOX2 to convert astrocytes to multipotent stem cells (Fig. 6). This highlights the importance of the cellular milieu on the reprogramming process.

Induced neuroblasts mature into synapse-forming interneurons

As DCX is restricted to neuroblasts and immature neurons and TUBB3 is also broadly expressed in both immature and mature neurons, the expression of these two markers during SOX2-induced neurogenesis does not indicate that these new neurons can become mature. In comparison, MAP2 and NeuN are markers for mature neurons. These markers were undetected in both BrdU- and tdT-traced cells in mGfap-CreRosa-tdT mice that were injected with SOX2-expressing virus at 4 wpi (Fig. 8a,f,g). When examined at 8 wpi, however,

3% of tdT + cells were labelled by either MAP2 or NeuN around the virus-injected region (Fig. 8b,c,f). Correspondingly, an estimated 1% of either MAP2- or NeuN-positive cells within the injected region also incorporated BrdU (Fig. 8d,e,g), indicating that they were mature neurons reprogrammed through cell division. VPA is a histone deacetylase inhibitor that enhances cellular reprogramming 43 and promotes normal neurogenesis and maturation of induced adult neuroblasts in the adult brain 29,44 . Here, we examined the effect of VPA on SOX2-induced neurogenesis in the adult spinal cord. Interestingly, 4 weeks of intraperitoneal injection of VPA (100 mg kg −1 , twice daily) resulted in nearly a twofold increase of tdT + cells being labelled by either MAP2 or NeuN in mGfap-CreRosa-tdT mice (Fig. 8f). These neurons could survive up to 30 wpi, the longest time examined (Supplementary Fig. 8). Quantification of BrdU-labelled cells also showed a threefold increase of newly generated MAP2 + or NeuN + mature neurons (Fig. 8g). Together, these data indicate that SOX2-induced neurons become mature by 8 wpi and can be further enhanced by treatment with VPA.

(a) Experimental scheme. mGfap-CreRosa-tdT mice were injected with hGFAP-SOX2 or a control virus immediately after injury and analysed by IHC at 4 (I) or 8 (II, III) wpi. (b,c) Expression of the mature neuronal markers MAP2 (b) or NeuN (c) in tdT + cells in SOX2 virus-injected spinal cords at 8 wpi. tdT-traced MAP2 + or NeuN + cells were not detectable in control virus-injected spinal cords. Orthogonal views of cells with expression of the indicated markers are also shown. Compared with endogenous spinal motoneurons (indicated by an asterisk in c), the SOX2-induced neurons are interneuron-like with a smaller soma (indicated by an arrow in c). (d,e) SOX2-induced mature neurons pass through a proliferative stage. Mice were treated with BrdU at 3–18 dpi and analysed at 8 wpi. Orthogonal views of BrdU-traced mature neurons are also shown. (f,g) Quantification of SOX2-induced mature neurons in the injured adult spinal cord (mean+s.d. n=5 mice per group *P<0.01 by Student’s t-test). Scale bar, 20 μm (be).

We analysed the cellular identity of the reprogrammed neurons in mGfap-CreRosa-tdT mice that were injected with hGFAP-SOX2 virus and treated with VPA for 4 weeks beginning at 4 wpi (Fig. 9a). These mice were also treated with BrdU from 3–18 dpi to label newly generated cells (Fig. 9a). Immunohistochemistry showed that none of the tdT + cells expressed choline acetyltransferase (ChAT), a marker for cholinergic motor neurons (Supplementary Fig. 9a). In contrast, the astrocyte-derived mature neurons (indicated by the expression of tdT and MAP2) were positive for GABA or vGLUT1, markers for inhibitory or excitatory neurons, respectively (Fig. 9b and Supplementary Fig. 9b,c). Importantly, co-staining with GABA and BrdU confirmed that these cells were indeed newly reprogrammed (Fig. 9c). The GABAergic neuronal identity was further demonstrated by staining with an antibody against glutamate decarboxylase (GAD65) (Fig. 9d). Interestingly, some tdT-traced cells were co-labelled by synapsin-1 (SYN1), a marker for presynaptic terminals, in spinal cords injected with SOX2 virus but not with a control virus (Fig. 9e). Confocal analyses under higher magnifications showed that dense bouton-like terminals co-stained with SYN1 and tdT were juxtaposed to the soma and axon of ChAT + cells (Fig. 9f,g), indicating the formation of synapses between SOX2-induced new neurons and local motor neurons. Together, these results showed that ectopic SOX2 could convert local astrocytes into synapse-forming GABAergic interneurons in the adult spinal cord.

(a) Experimental scheme to examine SOX2-induced mature neurons. (b,c) Ectopic SOX2 converts astrocytes (indicated by tdT) to GABAergic interneurons (indicated by GABA and GAD65 expression). Coexpression of the indicated markers is also shown by an orthogonal view. These cells were not detectable in spinal cords injected with a control virus. (d) BrdU labelling indicates that SOX2-induced GABA + cells are newly generated. Coexpression of the indicated markers is shown by an orthogonal view. (e) Expression of synapsin-I (SYN1) in tdT + cells in spinal cords injected with SOX2 but not a control virus. An orthogonal view is to show co-localization of the indicated markers. (f,g) Confocal images showing synapse-formation between SOX2-induced neurons (labelled by tdT) and the soma (f) or axon (g) of endogenous cholinergic motoneurons (labelled by ChAT). Synapses are indicated by SYN1-expression. Scale bar, 20 μm (bf) and 10 μm (g).


Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes

The quality of genetically encoded calcium indicators (GECIs) has improved dramatically in recent years, but high-performing ratiometric indicators are still rare. Here we describe a series of fluorescence resonance energy transfer (FRET)-based calcium biosensors with a reduced number of calcium binding sites per sensor. These 'Twitch' sensors are based on the C-terminal domain of Opsanus troponin C. Their FRET responses were optimized by a large-scale functional screen in bacterial colonies, refined by a secondary screen in rat hippocampal neuron cultures. We tested the in vivo performance of the most sensitive variants in the brain and lymph nodes of mice. The sensitivity of the Twitch sensors matched that of synthetic calcium dyes and allowed visualization of tonic action potential firing in neurons and high resolution functional tracking of T lymphocytes. Given their ratiometric readout, their brightness, large dynamic range and linear response properties, Twitch sensors represent versatile tools for neuroscience and immunology.


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