SOX2 and SOX9 Expression in Developing Postnatal Opossum (Monodelphis domestica) Cortex.

(1) Background: Central nervous system (CNS) development is characterized by dynamic changes in cell proliferation and differentiation. Key regulators of these transitions are the transcription factors such as SOX2 and SOX9. SOX2 is involved in the maintenance of progenitor cell state and neural stem cell multipotency, while SOX9, expressed in neurogenic niches, plays an important role in neuron/glia switch with predominant expression in astrocytes in the adult brain. (2) Methods: To validate SOX2 and SOX9 expression patterns in developing opossum (Monodelphis domestica) cortex, we used immunohistochemistry (IHC) and the isotropic fractionator method on fixed cortical tissue from comparable postnatal ages, as well as dissociated primary neuronal cultures. (3) Results: Neurons positive for both neuronal (TUJ1 or NeuN) and stem cell (SOX2) markers were identified, and their presence was confirmed with all methods and postnatal age groups (P4-6, P6-18, and P30) analyzed. SOX9 showed exclusive staining in non-neuronal cells, and it was coexpressed with SOX2. (4) Conclusions: The persistence of SOX2 expression in developing cortical neurons of M. domestica during the first postnatal month implies the functional role of SOX2 during neuronal differentiation and maturation, which was not previously reported in opossums.

Label-Free Long-Term Methods for Live Cell Imaging of Neurons: New Opportunities.

Time-lapse light microscopy combined with in vitro neuronal cultures has provided a significant contribution to the field of Developmental Neuroscience. The establishment of the neuronal polarity, i.e., formation of axons and dendrites, key structures responsible for inter-neuronal signaling, was described in 1988 by Dotti, Sullivan and Banker in a milestone paper that continues to be cited 30 years later. In the following decades, numerous fluorescently labeled tags and dyes were developed for live cell imaging, providing tremendous advancements in terms of resolution, acquisition speed and the ability to track specific cell structures. However, long-term recordings with fluorescence-based approaches remain challenging because of light-induced phototoxicity and/or interference of tags with cell physiology (e.g., perturbed cytoskeletal dynamics) resulting in compromised cell viability leading to cell death. Therefore, a label-free approach remains the most desirable method in long-term imaging of living neurons. In this paper we will focus on label-free high-resolution methods that can be successfully used over a prolonged period. We propose novel tools such as scanning ion conductance microscopy (SICM) or digital holography microscopy (DHM) that could provide new insights into live cell dynamics during neuronal development and regeneration after injury.

The Potential Connection between Molecular Changes and Biomarkers Related to ALS and the Development and Regeneration of CNS.

Neurodegenerative diseases are one of the greatest medical burdens of the modern age, being mostly incurable and with limited prognostic and diagnostic tools. Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease characterized by the loss of motoneurons, with a complex etiology, combining genetic, epigenetic, and environmental causes. The neuroprotective therapeutic approaches are very limited, while the diagnostics rely on clinical examination and the exclusion of other diseases. The recent advancement in the discovery of molecular pathways and gene mutations involved in ALS has deepened the understanding of the disease pathology and opened the possibility for new treatments and diagnostic procedures. Recently, 15 risk loci with distinct genetic architectures and neuron-specific biology were identified as linked to ALS through common and rare variant association analyses. Interestingly, the quantity of related proteins to these genes has been found to change during early postnatal development in mammalian spinal cord tissue (opossum Monodelphis domestica) at the particular time when neuroregeneration stops being possible. Here, we discuss the possibility that the ALS-related genes/proteins could be connected to neuroregeneration and development. Moreover, since the regulation of gene expression in developmental checkpoints is frequently regulated by non-coding RNAs, we propose that studying the changes in the composition and quantity of non-coding RNA molecules, both in ALS patients and in the developing central nervous (CNS) system of the opossum at the time when neuroregeneration ceases, could reveal potential biomarkers useful in ALS prognosis and diagnosis.

The Role of ATF3 in Neuronal Differentiation and Development of Neuronal Networks in Opossum Postnatal Cortical Cultures.

Activating transcription factor 3 (ATF3), a member of the ATF/cAMP response element-binding (CREB) family, is upregulated by various intracellular and extracellular signals such as injury and signals related to cell proliferation. ATF3 also belongs to the regeneration-associated genes (RAG) group of transcription factors. RAG and ATF/CREB transcription factors that play an important role in embryonic neuronal development and PNS regeneration may also be involved in postnatal neuronal differentiation and development, as well as in the regeneration of the injured CNS. Here we investigated the effect of ATF3 in differentiation, neural outgrowth, network formation, and regeneration after injury using postnatal dissociated cortical neurons derived from neonatal opossums (Monodelphis domestica). Our results show that RAG and ATF genes are differentially expressed in early differentiated neurons versus undifferentiated neurospheres and that many members of those families, ATF3 in particular, are upregulated in cortical cultures obtained from younger animals that have the ability to fully functionally regenerate spinal cord after injury. In addition, we observed different intracellular localization of ATF3 that shifts from nuclear (in neuronal progenitors) to cytoplasmic (in more mature neurons) during neuronal differentiation. The ATF3 inhibition, pharmacological or by specific antibody, reduced the neurite outgrowth and differentiation and caused increased cell death in early differentiating cortical neuronal cultures, suggesting the importance of ATF3 in the CNS development of neonatal opossums. Finally, we investigated the regeneration capacity of primary cortical cultures after mechanical injury using the scratch assay. Remarkably, neonatal opossum-derived cultures retain their capacity to regenerate for up to 1 month in vitro. Inhibition of ATF3 correlates with reduced neurite outgrowth and regeneration after injury. These results indicate that ATF3, and possibly other members of RAG and ATF/CREB family of transcription factors, have an important role both during cortical postnatal development and in response after injury.

Proteomic analysis of opossum Monodelphis domestica spinal cord reveals the changes of proteins related to neurodegenerative diseases during developmental period when neuroregeneration stops being possible.

One of the major challenges of modern neurobiology concerns the inability of the adult mammalian central nervous system (CNS) to regenerate and repair itself after injury. It is still unclear why the ability to regenerate CNS is lost during evolution and development and why it becomes very limited in adult mammals. A convenient model to study cellular and molecular basis of this loss is neonatal opossum (Monodelphis domestica). Opossums are marsupials that are born very immature with the unique possibility to successfully regenerate postnatal spinal cord after injury in the first two weeks of their life, after which this ability abbruptly stops. Using comparative proteomic approach we identified the proteins that are differentially distributed in opossum spinal tissue that can and cannot regenerate after injury, among which stand out the proteins related to neurodegenerative diseases (NDD), such as Huntington, Parkinson and Alzheimer's disease, previously detected by comparative transcriptomics on the analog tissue. The different distribution of the selected proteins detected by comparative proteomics was further confirmed by Western blot (WB), and the changes in the expression of related genes were analysed by quantitative reverse transcription PCR (qRT-PCR). Furthermore, we explored the cellular localization of the selected proteins using immunofluorescent microscopy. To our knowledge, this is the first report on proteins differentially present in developing, non-injured mammalian spinal cord tissue with different regenerative capacities. The results of this study indicate that the proteins known to have an important role in the pathophysiology of neurodegeneration in aged CNS, could also have an important phyisological role during CNS postnatal development and in neuroregeneration process.

Establishment of Long-Term Primary Cortical Neuronal Cultures From Neonatal Opossum Monodelphis domestica.

Primary dissociated neuronal cultures have become a standard model for studying central nervous system (CNS) development. Such cultures are predominantly prepared from the hippocampus or cortex of rodents (mice and rats), while other mammals are less used. Here, we describe the establishment and extensive characterization of the primary dissociated neuronal cultures derived from the cortex of the gray South American short-tailed opossums, Monodelphis domestica. Opossums are unique in their ability to fully regenerate their CNS after an injury during their early postnatal development. Thus, we used cortex of postnatal day (P) 3-5 opossum to establish long-surviving and nearly pure neuronal cultures, as well as mixed cultures composed of radial glia cells (RGCs) in which their neurogenic and gliogenic potential was confirmed. Both types of cultures can survive for more than 1 month in vitro. We also prepared neuronal cultures from the P16-18 opossum cortex, which were composed of astrocytes and microglia, in addition to neurons. The long-surviving opossum primary dissociated neuronal cultures represent a novel mammalian in vitro platform particularly useful to study CNS development and regeneration.

Glia in amyotrophic lateral sclerosis and spinal cord injury: common therapeutic targets.

The toolkit for repairing damaged neurons in amyotrophic lateral sclerosis (ALS) and spinal cord injury (SCI) is extremely limited. Here, we reviewed the in vitro and in vivo studies and clinical trials on nonneuronal cells in the neurodegenerative processes common to both these conditions. Special focus was directed to microglia and astrocytes, because their activation and proliferation, also known as neuroinflammation, is a key driver of neurodegeneration. Neuroinflammation is a multifaceted process that evolves during the disease course, and can be either beneficial or toxic to neurons. Given the fundamental regulatory functions of glia, pathogenic mechanisms in neuroinflammation represent promising therapeutic targets. We also discussed neuroprotective, immunosuppressive, and stem-cell based approaches applicable to both ALS and SCI.

Improved neuron culture using scaffolds made of three-dimensional PDMS micro-lattices.

Tissue engineering strives to create functional components of organs with different cell types in vitro. One of the challenges is to fabricate scaffolds for three-dimensional (3D) cell culture under physiological conditions. Of particular interest is the investigation of the morphology and function of the central nervous system cultured using such scaffolds. Here, we used an elastomer-polydimethylsiloxane (PDMS)-to produce lattice-type scaffolds from a photolithography-defined template. The photomask with antidot arrays was spin-coated by a thick layer of resist, and was downward mounted on a rotating stage at an angle of 45°. After the exposure was repeated three or more times, maintaining the same exposure plan but rotated by the same angle, a photoresist was developed to produce a 3D porous template. Afterwards, a pre-polymer mixture of PDMS was poured in and cured, followed by a resist etch, resulting in lattice-type PDMS features. Before cell culture, the PDMS lattices were surface functionalized. A culture test was conducted using NIH-3T3 cells and primary hippocampal cells from rats, showing homogenous cell infiltration and 3D attachment. As expected, a much higher cell number was found in the 3D PDMS lattices compared to the 2D culture. We also found a higher neuron-to-astrocyte ratio and a higher degree of cell ramification in the 3D culture compared to the 2D culture due to the change of scaffold topography and the elastic properties of the PDMS micro-lattices. Our results demonstrate that the 3D PDMS micro-lattices improve the survival and growth of cells, as well as the network formation of neurons. We believe that such an enabling technology is useful for research and clinical applications, including disease modeling, regenerative medicine, and drug discovery/drug cytotoxicity studies.

An improved method for growing neurons: Comparison with standard protocols.

BACKGROUND: Since different culturing parameters - such as media composition or cell density - lead to different experimental results, it is important to define the protocol used for neuronal cultures. The vital role of astrocytes in maintaining homeostasis of neurons - both in vivo and in vitro - is well established: the majority of improved culturing conditions for primary dissociated neuronal cultures rely on astrocytes. NEW METHOD: Our culturing protocol is based on a novel serum-free preparation of astrocyte - conditioned medium (ACM). We compared the proposed ACM culturing method with other two commonly used methods Neurobasal/B27- and FBS- based media. We performed morphometric characterization by immunocytochemistry and functional analysis by calcium imaging for all three culture methods at 1, 7, 14 and 60days in vitro (DIV). RESULTS: ACM-based cultures gave the best results for all tested criteria, i.e. growth cone's size and shape, neuronal outgrowth and branching, network activity and synchronization, maturation and long-term survival. The differences were more pronounced when compared with FBS-based medium. Neurobasal/B27 cultures were comparable to ACM for young cultures (DIV1), but not for culturing times longer than DIV7. COMPARISON WITH EXISTING METHOD(S): ACM-based cultures showed more robust neuronal outgrowth at DIV1. At DIV7 and 60, the activity of neuronal network grown in ACM had a more vigorous spontaneous electrical activity and a higher degree of synchronization. CONCLUSIONS: We propose our ACM-based culture protocol as an improved and more suitable method for both short- and long-term neuronal cultures.

High aspect ratio silicon nanowires control fibroblast adhesion and cytoskeleton organization.

Cell-cell and cell-matrix interactions are essential to the survival and proliferation of most cells, and are responsible for triggering a wide range of biochemical pathways. More recently, the biomechanical role of those interactions was highlighted, showing, for instance, that adhesion forces are essential for cytoskeleton organization. Silicon nanowires (Si NWs) with their small size, high aspect ratio and anisotropic mechanical response represent a useful model to investigate the forces involved in the adhesion processes and their role in cellular development. In this work we explored and quantified, by single cell force spectroscopy (SCFS), the interaction of mouse embryonic fibroblasts with a flexible forest of Si NWs. We observed that the cell adhesion forces are comparable to those found on collagen and bare glass coverslip, analogously the membrane tether extraction forces are similar to that on collagen but stronger than that on bare flat glass. Cell survival did not depend significantly on the substrate, although a reduced proliferation after 36 h was observed. On the contrary both cell morphology and cytoskeleton organization revealed striking differences. The cell morphology on Si-NW was characterized by a large number of filopodia and a significant decrease of the cell mobility. The cytoskeleton organization was characterized by the absence of actin fibers, which were instead dominant on collagen and flat glass support. Such findings suggest that the mechanical properties of disordered Si NWs, and in particular their strong asymmetry, play a major role in the adhesion, morphology and cytoskeleton organization processes. Indeed, while adhesion measurements by SCFS provide out-of-plane forces values consistent with those measured on conventional substrates, weaker in-plane forces hinder proper cytoskeleton organization and migration processes.

The role of dimensionality in neuronal network dynamics.

Recent results from network theory show that complexity affects several dynamical properties of networks that favor synchronization. Here we show that synchronization in 2D and 3D neuronal networks is significantly different. Using dissociated hippocampal neurons we compared properties of cultures grown on a flat 2D substrates with those formed on 3D graphene foam scaffolds. Both 2D and 3D cultures had comparable glia to neuron ratio and the percentage of GABAergic inhibitory neurons. 3D cultures because of their dimension have many connections among distant neurons leading to small-world networks and their characteristic dynamics. After one week, calcium imaging revealed moderately synchronous activity in 2D networks, but the degree of synchrony of 3D networks was higher and had two regimes: a highly synchronized (HS) and a moderately synchronized (MS) regime. The HS regime was never observed in 2D networks. During the MS regime, neuronal assemblies in synchrony changed with time as observed in mammalian brains. After two weeks, the degree of synchrony in 3D networks decreased, as observed in vivo. These results show that dimensionality determines properties of neuronal networks and that several features of brain dynamics are a consequence of its 3D topology.

Can hippocampal neurites and growth cones climb over obstacles?

Guidance molecules, such as Sema3A or Netrin-1, can induce growth cone (GC) repulsion or attraction in the presence of a flat surface, but very little is known of the action of guidance molecules in the presence of obstacles. Therefore we combined chemical and mechanical cues by applying a steady Netrin-1 stream to the GCs of dissociated hippocampal neurons plated on polydimethylsiloxane (PDMS) surfaces patterned with lines 2 µm wide, with 4 µm period and with a height varying from 100 to 600 nm. GC turning experiments performed 24 hours after plating showed that filopodia crawl over these lines within minutes. These filopodia do not show staining for the adhesion marker Paxillin. GCs and neurites crawl over lines 100 nm high, but less frequently and on a longer time scale over lines higher than 300 nm; neurites never crawl over lines 600 nm high. When neurons are grown for 3 days over patterned surfaces, also neurites can cross lines 300 nm and 600 nm high, grow parallel to and on top of these lines and express Paxillin. Axons - selectively stained with SMI 312 - do not differ from dendrites in their ability to cross these lines. Our results show that highly motile structures such as filopodia climb over high obstacle in response to chemical cues, but larger neuronal structures are less prompt and require hours or days to climb similar obstacles.

Comparison of the force exerted by hippocampal and DRG growth cones.

Mechanical properties such as force generation are fundamental for neuronal motility, development and regeneration. We used optical tweezers to compare the force exerted by growth cones (GCs) of neurons from the Peripheral Nervous System (PNS), such as Dorsal Root Ganglia (DRG) neurons, and from the Central Nervous System (CNS) such as hippocampal neurons. Developing GCs from dissociated DRG and hippocampal neurons were obtained from P1-P2 and P10-P12 rats. Comparing their morphology, we observed that the area of GCs of hippocampal neurons was 8-10 µm(2) and did not vary between P1-P2 and P10-P12 rats, but GCs of DRG neurons were larger and their area increased from P1-P2 to P10-P12 by 2-4 times. The force exerted by DRG filopodia was in the order of 1-2 pN and never exceeded 5 pN, while hippocampal filopodia exerted a larger force, often in the order of 5 pN. Hippocampal and DRG lamellipodia exerted lateral forces up to 20 pN, but lamellipodia of DRG neurons could exert a vertical force larger than that of hippocampal neurons. Force-velocity relationships (Fv) in both types of neurons had the same qualitative behaviour, consistent with a common autocatalytic model of force generation. These results indicate that molecular mechanisms of force generation of GC from CNS and PNS neurons are similar but the amplitude of generated force is influenced by their cytoskeletal properties.

Nanomechanics controls neuronal precursors adhesion and differentiation.

The ability to control the differentiation of stem cells into specific neuronal types has a tremendous potential for the treatment of neurodegenerative diseases. In vitro neuronal differentiation can be guided by the interplay of biochemical and biophysical cues. Different strategies to increase the differentiation yield have been proposed, focusing everything on substrate topography, or, alternatively on substrate stiffness. Both strategies demonstrated an improvement of the cellular response. However it was often impossible to separate the topographical and the mechanical contributions. Here we investigate the role of the mechanical properties of nanostructured substrates, aiming at understanding the ultimate parameters which govern the stem cell differentiation. To this purpose a set of different substrates with controlled stiffness and with or without nanopatterning are used for stem cell differentiation. Our results show that the neuronal differentiation yield depends mainly on the substrate mechanical properties while the geometry plays a minor role. In particular nanostructured and flat polydimethylsiloxane (PDMS) substrates with comparable stiffness show the same neuronal yield. The improvement in the differentiation yield obtained through surface nanopatterning in the submicrometer scale could be explained as a consequence of a substrate softening effect. Finally we investigate by single cell force spectroscopy the neuronal precursor adhesion on the substrate immediately after seeding, as a possible critical step governing the neuronal differentiation efficiency. We observed that neuronal precursor adhesion depends on substrate stiffness but not on surface structure, and in particular it is higher on softer substrates. Our results suggest that cell-substrate adhesion forces and mechanical response are the key parameters to be considered for substrate design in neuronal regenerative medicine.

Less than 5 Netrin-1 molecules initiate attraction but 200 Sema3A molecules are necessary for repulsion.

Guidance molecules, such as Sema3A or Netrin-1, induce growth cone (GC) repulsion or attraction. In order to determine the speed of action and efficiency of these guidance cues we developed an experimental procedure to deliver controlled amounts of these molecules. Lipid vesicles encapsulating 10-10(4) molecules of Sema3A or Netrin-1 were manipulated with high spatial and temporal resolution by optical tweezers and their photolysis triggered by laser pulses. Guidance molecules released from the vesicles diffused and reached the GC membrane in a few seconds. Following their arrival, GCs retracted or grew in 20-120 s. By determining the number of guidance molecules trapped inside vesicles and estimating the fraction of guidance molecules reaching the GC, we show that the arrival of less than 5 Netrin-1 molecules on the GC membrane is sufficient to induce growth. In contrast, the arrival of about 200 Sema3A molecules is necessary to induce filopodia repulsion.

Acceleration of neuronal precursors differentiation induced by substrate nanotopography.

Embryonic stem (ES) cell differentiation in specific cell lineages is a major issue in cell biology particularly in regenerative medicine. Differentiation is usually achieved by using biochemical factors and it is not clear whether mechanical properties of the substrate over which cells are grown can affect proliferation and differentiation. Therefore, we produced patterns in polydimethylsiloxane (PDMS) consisting of groove and pillar arrays of sub-micrometric lateral resolution as substrates for cell cultures. We analyzed the effect of different nanostructures on differentiation of ES-derived neuronal precursors into neuronal lineage without adding biochemical factors. Neuronal precursors adhered on PDMS more effectively than on glass coverslips. We demonstrated that neuronal yield was enhanced by increasing pillars height from 35 to 400 nm. On higher pillar neuronal differentiation reaches ∼80% 96 h after plating and the largest differentiation enhancement of pillars over flat PDMS was observed during the first 6 h of culture. We conclude that PDMS nanopillars accelerate and increase neuronal differentiation.

Fragmentation as a mechanism for growth cone pruning and degeneration.

During early development of the central nervous system, there is an excessive outgrowth of neuronal projections, which later need to be refined to achieve precise connectivity. Axon pruning and degeneration are strategies used to remove exuberant neurites and connections in the immature nervous system to ensure the proper formation of functional circuitry. To observe morphological changes and physical mechanisms underlying this process, early differentiating embryonic stem cell-derived neurons were used combining video imaging of live growth cones (GCs) with confocal laser scanning microscopy and atomic force microscopy, both on fixed and living neurons. Using this method, we could highlight the presence of submicrometric fragments in still and in some of the retracting GCs. The observed fragmentation is not an artifact of atomic force microscopy scanning or fixation, or the result of apoptosis. Therefore, the morphology of GCs depends on their overall motility, and fragmentation seems to be the fate of GCs that have not found a correct destination.

Integration of confocal and atomic force microscopy images.

Atomic force microscopy (AFM) provides the possibility to map the 3D structure of viewed objects with a nanometric resolution, which cannot be achieved with other imaging methods such as conventional video imaging and confocal fluorescent microscopy. Video imaging with CCD cameras can provide an analysis of biological events with a temporal and spatial resolution not possible with AFM, while confocal imaging allows the simultaneous acquisition of immunofluorescence images. In this communication we present a simple method to combine AFM and confocal images to study differentiating embryonic stem (ES) cells-derived and dorsal root ganglia (DRG) neurons in culture. Neurons were grown on coverslips with micrometric markers that allow finding and imaging the same neuron with different microscopes. AFM and confocal images were registered using conventional methods used in Computer Science. The combination of these two techniques allows relating functional properties to morphological features of imaged neurons.

Embryonic stem cell-derived neurons form functional networks in vitro.

Embryonic stem (ES) cells provide a flexible and unlimited source for a variety of neuronal types. Because mature neurons establish neuronal networks very easily, we tested whether ES-derived neurons are capable of generating functional networks and whether these networks, generated in vitro, are capable of processing information. Single-cell electrophysiology with pharmacological antagonists demonstrated the presence of both excitatory and inhibitory synaptic connections. Extracellular recording with planar multielectrode arrays showed that spontaneous bursts of electrical activity are present in ES-derived networks with properties remarkably similar to those of hippocampal neurons. When stimulated with extracellular electrodes, ES-derived neurons fired action potentials, and the evoked electrical activity spread throughout the culture. A statistical analysis indicated that ES-derived networks discriminated between stimuli of different intensity at a single trial level, a key feature for an efficient information processing. Thus, ES-derived neurons provide a novel in vitro strategy to create functional networks with defined computational properties.