Florentia Papastefanaki

Schwann cells engineered to express the cell adhesion molecule L1 accelerate myelination and motor recovery after spinal cord injury

Functional recovery after spinal cord lesion remains an important goal. A combination of inhibitory molecules and lack of appropriate permissive factors in the lesioned spinal cord results in failure of fiber tract reconnection and function. Experimental transplantation in rodent and primate models of CNS injuries has led to the idea that Schwann cells (SCs) are promising candidates for autologous transplantation to assist myelination of lesions and to deliver therapeutic agents in the CNS. In this study, we used retroviral transduction to genetically modify SCs from transgenic GFP-mice in order to overexpress the cell adhesion molecule L1, a protein promoting neurite outgrowth and implicated in myelination. SCs transduced to express L1 or its chimeric secreted form L1-Fc were mixed and grafted rostrally to the lesion site of adult mice immediately after spinal cord compression injury. Our results indicate that 3 weeks postoperatively, but not thereafter, mice transplanted with L1/L1-Fc-expressing SCs exhibited faster locomotor recovery as compared to animals which received SCs transduced with a control vector or no cells at all. Morphological analysis indicated that the accelerated functional recovery correlated with earlier and enhanced myelination by both grafted and host SCs. Moreover, increased sprouting of serotonergic fibers into and across the lesion site was observed in the L1/L1-Fc group as compared with controls. Our results suggest that transplantation of L1-overexpressing SCs enhances early events in spinal cord repair after injury and may be considered in combinatorial strategies together with other regeneration-promoting molecules.

From demyelination to remyelination: the road toward therapies for spinal cord injury.

Myelin integrity is crucial for central nervous system (CNS) physiology while its preservation and regeneration after spinal cord injury (SCI) is key to functional restoration. Disturbance of nodal organization acutely after SCI exposes the axon and triggers conduction block in the absence of overt demyelination. Oligodendrocyte (OL) loss and myelin degradation follow as a consequence of secondary damage. Here, we provide an overview of the major biological events and underlying mechanisms leading to OL death and demyelination and discuss strategies to restrain these processes. Another aspect which is critical for SCI repair is the enhancement of endogenously occurring spontaneous remyelination. Recent findings have unveiled the complex roles of innate and adaptive immune responses in remyelination and the immunoregulatory potential of the glial scar. Moreover, the intimate crosstalk between neuronal activity, oligodendrogenesis and myelination emphasizes the contribution of rehabilitation to functional recovery. With a view toward clinical applications, several therapeutic strategies have been devised to target SCI pathology, including genetic manipulation, administration of small therapeutic molecules, immunomodulation, manipulation of the glial scar and cell transplantation. The implementation of new tools such as cellular reprogramming for conversion of one somatic cell type to another or the use of nanotechnology and tissue engineering products provides additional opportunities for SCI repair. Given the complexity of the spinal cord tissue after injury, it is becoming apparent that combinatorial strategies are needed to rescue OLs and myelin at early stages after SCI and support remyelination, paving the way toward clinical translation. GLIA 2015;63:1101–1125

Cell Adhesion Molecules in Gene and Cell Therapy Approaches for Nervous System Repair

The inability of the central nervous system (CNS) to efficiently repair damages results in severe functional impairment after trauma or neurodegenerative/demyelinating diseases. Regeneration failure is attributed to inhibitory molecules creating a nonpermissive environment for axonal regrowth, and dictates the necessity for the development of novel therapeutic strategies. An emerging approach for improving regeneration is the use of gene therapy to manipulate cell adhesion molecule expression in experimental animal models of degeneration. Alternatively, cell transplantation to replace lost neurons and the grafting of myelinating cells to repair demyelinating lesions are promising approaches for treating CNS injuries and demyelination. Schwann cells (SCs), oligodendrocyte progenitors, olfactory ensheathing cells and embryonic and neural stem cells have been shown to form myelin after transplantation into the demyelinated CNS. The repair capacity of the peripheral nervous system (PNS) is much higher, but there is still a limit to the amount of nerve loss that can be bridged after injury, and longer nerve gaps call for the use of conduits populated with living cells. In both cases, the interaction of grafted cells with the host environment is of paramount importance for the incorporation and functional integration of these cells and the manipulation of cell adhesion molecules is an attractive approach towards achieving this goal. In this review we summarize data from the recent literature regarding the manipulation of cell adhesion molecule expression towards CNS and PNS repair and discuss the prospects for future therapeutic applications.

Functional Cross-Talk Between the Cellular Prion Protein and the Neural Cell Adhesion Molecule is Critical for Neuronal Differentiation of Neural Stem/Precursor Cells

Cellular prion protein (PrP) is prominently expressed in brain, in differentiated neurons but also in neural stem/precursor cells (NPCs). The misfolding of PrP is a central event in prion diseases, yet the physiological function of PrP is insufficiently understood. Although PrP has been reported to associate with the neural cell adhesion molecule (NCAM), the consequences of concerted PrP‐NCAM action in NPC physiology are unknown. Here, we generated NPCs from the subventricular zone (SVZ) of postnatal day 5 wild‐type and PrP null (−/−) mice and observed that PrP is essential for proper NPC proliferation and neuronal differentiation. Moreover, we found that PrP is required for the NPC response to NCAM‐induced neuronal differentiation. In the absence of PrP, NCAM not only fails to promote neuronal differentiation but also induces an accumulation of doublecortin‐positive neuronal progenitors at the proliferation stage. In agreement, we noted an increase in cycling neuronal progenitors in the SVZ of PrP−/− mice compared with PrP+/+ mice, as evidenced by double labeling for the proliferation marker Ki67 and doublecortin as well as by 5‐bromo‐2′‐deoxyuridine incorporation experiments. Additionally, fewer newly born neurons were detected in the rostral migratory stream of PrP−/− mice. Analysis of the migration of SVZ cells in microexplant cultures from wild‐type and PrP−/− mice revealed no differences between genotypes or a role for NCAM in this process. Our data demonstrate that PrP plays a critical role in neuronal differentiation of NPCs and suggest that this function is, at least in part, NCAM‐dependent. Stem Cells 2014;32:1674–1687

Nonyloxytryptamine mimics polysialic acid and modulates neuronal and glial functions in cell culture.

Polysialic acid (PSA) is a major regulator of cell–cell interactions in the developing nervous system and in neural plasticity in the adult. As a polyanionic molecule with high water‐binding capacity, PSA increases the intercellular space generating permissive conditions for cell motility. PSA enhances stem cell migration and axon path finding and promotes repair in the lesioned peripheral and central nervous systems, thus contributing to regeneration. As a next step in developing an improved PSA‐based approach to treat nervous system injuries, we searched for small organic compounds that mimic PSA and identified as a PSA mimetic 5‐nonyloxytryptamine oxalate, described as a selective 5‐hydroxytryptamine receptor 1B (5‐HT1B) agonist. Similar to PSA, 5‐nonyloxytryptamine binds to the PSA‐specific monoclonal antibody 735, enhances neurite outgrowth of cultured primary neurons and process formation of Schwann cells, protects neurons from oxidative stress, reduces migration of astrocytes and enhances myelination in vitro. Furthermore, nonyloxytryptamine treatment enhances expression of the neural cell adhesion molecule (NCAM) and its polysialylated form PSA‐NCAM and reduces expression of the microtubule‐associated protein MAP2 in cultured neuroblastoma cells. These results demonstrate that 5‐nonyloxytryptamine mimics PSA and triggers PSA‐mediated functions, thus contributing to the repertoire of molecules with the potential to improve recovery in acute and chronic injuries of the mammalian peripheral and central nervous systems.

Neural stem/progenitor cells differentiate into oligodendrocytes, reduce inflammation, and ameliorate learning deficits after transplantation in a mouse model of traumatic brain injury

The central nervous system has limited capacity for regeneration after traumatic injury. Transplantation of neural stem/progenitor cells (NPCs) has been proposed as a potential therapeutic approach while insulin‐like growth factor I (IGF‐I) has neuroprotective properties following various experimental insults to the nervous system. We have previously shown that NPCs transduced with a lentiviral vector for IGF‐I overexpression have an enhanced ability to give rise to neurons in vitro but also in vivo, upon transplantation in a mouse model of temporal lobe epilepsy. Here we studied the regenerative potential of NPCs, IGF‐I‐transduced or not, in a mouse model of hippocampal mechanical injury. NPC transplantation, with or without IGF‐I transduction, rescued the injury‐induced spatial learning deficits as revealed in the Morris Water Maze. Moreover, it had beneficial effects on the host tissue by reducing astroglial activation and microglial/macrophage accumulation while enhancing generation of endogenous oligodendrocyte precursor cells. One or two months after transplantation the grafted NPCs had migrated towards the lesion site and in the neighboring myelin‐rich regions. Transplanted cells differentiated toward the oligodendroglial, but not the neuronal or astrocytic lineages, expressing the early and late oligodendrocyte markers NG2, Olig2, and CNPase. The newly generated oligodendrocytes reached maturity and formed myelin internodes. Our current and previous observations illustrate the high plasticity of transplanted NPCs which can acquire injury‐dependent phenotypes within the host CNS, supporting the fact that reciprocal interactions between transplanted cells and the host tissue are an important factor to be considered when designing prospective cell‐based therapies for CNS degenerative conditions. GLIA 2016;64:763–779

Defective synaptic connectivity and axonal neuropathology in a human iPSC-based model of familial Parkinson’s disease

Significance Parkinson’s disease (PD) is an incurable neurodegenerative disorder characterized by motor and nonmotor deficits, including cognitive decline and dementia. The protein αSyn is strongly associated with PD pathogenesis, whereas αSyn mutations, such as p.A53T, cause familial forms of PD. Animal models are crucial for understanding PD pathogenesis, but there are limitations in the extent to which these models reproduce faithfully the human disease. Cell-reprogramming technologies allow the generation of human neurons from patients with PD, but it has proven difficult to identify cellular pathologies in induced pluripotent stem cell–derived neurons. In this study, we created a robust p.A53T patient–derived model of PD that captures disease-related phenotypes under basal conditions, thus providing a unique system for studies of disease mechanisms and development of therapeutics. α-Synuclein (αSyn) is the major gene linked to sporadic Parkinson’s disease (PD), whereas the G209A (p.A53T) αSyn mutation causes a familial form of PD characterized by early onset and a generally severe phenotype, including nonmotor manifestations. Here we generated de novo induced pluripotent stem cells (iPSCs) from patients harboring the p.A53T mutation and developed a robust model that captures PD pathogenic processes under basal conditions. iPSC-derived mutant neurons displayed novel disease-relevant phenotypes, including protein aggregation, compromised neuritic outgrowth, and contorted or fragmented axons with swollen varicosities containing αSyn and Tau. The identified neuropathological features closely resembled those in brains of p.A53T patients. Small molecules targeting αSyn reverted the degenerative phenotype under both basal and induced stress conditions, indicating a treatment strategy for PD and other synucleinopathies. Furthermore, mutant neurons showed disrupted synaptic connectivity and widespread transcriptional alterations in genes involved in synaptic signaling, a number of which have been previously linked to mental disorders, raising intriguing implications for potentially converging disease mechanisms.

Intraspinal Delivery of Polyethylene Glycol-coated Gold Nanoparticles Promotes Functional Recovery After Spinal Cord Injury

Failure of the mammalian central nervous system (CNS) to regenerate effectively after injury leads to mostly irreversible functional impairment. Gold nanoparticles (AuNPs) are promising candidates for drug delivery in combination with tissue-compatible reagents, such as polyethylene glycol (PEG). PEG administration in CNS injury models has received interest for potential therapy, but toxicity and low bioavailability prevents clinical application. Here we show that intraspinal delivery of PEG-functionalized 40-nm-AuNPs at early stages after mouse spinal cord injury is beneficial for recovery. Positive outcome of hind limb motor function was accompanied by attenuated inflammatory response, enhanced motor neuron survival, and increased myelination of spared or regrown/sprouted axons. No adverse effects, such as body weight loss, ill health, or increased mortality were observed. We propose that PEG-AuNPs represent a favorable drug-delivery platform with therapeutic potential that could be further enhanced if PEG-AuNPs are used as carriers of regeneration-promoting molecules.

Soluble forms of the cell adhesion molecule L1 produced by insect and baculovirus‐transduced mammalian cells enhance Schwann cell motility

J. Neurochem. (2010) 115, 1137–1149.

BM88/Cend1 regulates stimuli-induced intracellular calcium mobilization

In neurogenesis, little is known about signal transduction pathways upstream of gene expression however, mounting evidence suggests that calcium release from internal stores plays a critical role. We have previously demonstrated that BM88 is a neuronal lineage-specific regulator of cell cycle exit and differentiation; we now report a link between BM88 and calcium signaling. Calcium imaging experiments revealed that P2Y-induced calcium mobilization is diminished in mouse neuroblastoma Neuro 2a cells stably transfected with BM88 (N2A-BM88 cells) as compared with N2A cells or N2A cells differentiated with retinoic acid. This effect is not restricted to N2A cells but is also observed in HeLa cells that are transiently transfected with BM88, indicating that cells of both neural and non-neural origin respond similarly. Further, activation of P2Y1 but not purinergic P2X receptors induces proliferation of N2A and to a lesser extent of N2A-BM88 cells. Conversely, knockdown of BM88 facilitates N2A cell proliferation both under stimulating and non-stimulating conditions. Importantly, N2A-BM88 cells are less susceptible to apoptosis triggered by C2-ceramide and exhibit reduced C2-ceramide-induced intracellular calcium release. Higher calcium uptake from mitochondria and/or lower calcium levels inside the endoplasmic reticulum may explain the reduced calcium mobilization in response to BM88. Overall, our data reveal a novel signaling mechanism by which BM88 interferes with calcium release from inositol 1,4,5-trisphosphate-sensitive stores and exerts anti-proliferative and anti-apoptotic functions.