Peripheral nerve injuries occur with high incidence and often result in profound and permanent impact on the life of patients and on healthcare expenditure. Schwann cells (SC) play a promoting role in peripheral nerve regeneration providing physical and neurotrophic support that aids axon re-growth. However, these beneficial properties are not exploitable in nerve tissue engineering due to the difficulties in SC harvesting and expansion in culture. Adult stem cells derived from bone marrow (BM-MSC) and from adipose tissue (ASC) can be differentiated in SC-like cells and be used as SC substitutes in bioengineered nerve conduits for the improvement of peripheral nerve regeneration. Pharmacological intervention approaches for the treatment of nerve injury are still not clinically available. Nevertheless, γ-Aminobutyric acid (GABA) receptors have been recently suggested as a putative target for such purpose. GABA is the main inhibitory neurotransmitter of the adult brain and interacts with two different receptor types. However, both GABA-A and GABA-B receptor types are functionally expressed also in SC, where they are involved in the regulation of SC physiology and in the development of the peripheral nervous system (PNS).The aim of this thesis was to characterise the GABAergic system of BM-MSC and ASC differentiated into a SC-like phenotype and to evaluate changes in the expression levels following differentiation. Moreover, the effect of specific GABA receptor ligands on cell proliferation and neurotrophic potential of differentiated stem cells were assessed. Using reverse transcriptase polymerase chain reaction, western blot analysis and immunohistochemistry we demonstrated that adult stem cells express several subunits of both GABA-A and GABA-B receptor systems such as GABA-B1a, GABA-B1b and GABA-B2, as well as GABA-A α2 and GABA-A β3. Expression levels and cellular localisation were comparable with adult and neonatal SC cultures used as positive controls, and protein expression levels for some of the subunits changed following glial differentiation. Interestingly, stimulation of GABA receptors with specific agonists influenced stem cell proliferation in two opposite ways. Baclofen, a GABA-B receptor agonist decreased proliferation of SC and differentiated ASC (dASC), but not of SC-like BM-MSC (dBM-MSC). By contrast, muscimol, a GABA-A receptor agonist, increased proliferation in SC and in both dASC and dBM-MSC. This suggests that GABAergic signalling could be a potential player in the mechanisms regulating stem cell differentiation and proliferation as reported in SC. Finally, baclofen treatments on SC and dASC modulated the expression levels and the release of the neurotrophins BDNF and NGF, which are key actors in the processes involved with peripheral nerve regeneration. Although further studies will be needed to clarify the role of GABA receptors in the PNS, the presence of functional GABA receptors on SC-like adult stem cells could represent an exploitable pharmacological target to modulate stem cell physiology and improve their neurotrophic potential for peripheral nerve regeneration.

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In der Entwicklung des peripheren Nervensystems formen Schwannzellen eine Myelinscheide um Axone mit einem Durchmesser von mehr als 1 μm durch die Bildung multipler kompakter Membranschichten. Voraussetzung einer optimalen Nervenleitgeschwindigkeit ist dabei ein physiologisches Verhältnis der Dicke der Myelinscheide zu dem jeweiligen Axondurchmesser. Eine zentrale Rolle spielt dabei der axonale EGF-like growth factor NRG1 Typ III, der ErbB2/3- Rezeptoren der Schwannzelle bindet. Der PI3K-AKT-Signalweg ist ein bekannter intrazellulärer Effektor des ErbB2/3-Rezeptors und wurde bereits mit dem Prozess der Myelinisierung in Verbindung gebracht. Um die spezifische Funktion des PI3K-AKT-Signalwegs in Schwannzellen zu erforschen, generierten wir mit Hilfe des Cre/LoxP-Systems Mausmutanten, die eine zellspezifische Inaktivierung des Gens Phosphatase and Tensin Homolog (Pten) in myelinisierenden Gliazellen aufweisen (Pten-Mutanten). Der Verlust der Lipidphosphatase PTEN führte zu einer Anreicherung ihres Substrates, des second messenger Phosphatidyl-(3,4,5)-Trisphosphat (PIP3), und damit zu einer gesteigerten Aktivität des PI3K-AKT-Signalwegs in den Schwannzellen der Pten-Mutanten. Wir beobachteten in den Pten-Mutanten eine ektopische Myelinisierung von unmyelinisierten C- Faser-Axonen sowie eine Hypermyelinisierung von Axonen bis 2 μm Durchmesser. Bei Axonen über 2 μm Durchmesser kam es zu Myelinausfaltungen und fokalen Hypermyelinisierungen (Tomacula) anliegend an Regionen des unkompakten Myelins (Paranodien und Schmidt- Lantermann-Inzisuren). Weiterhin bildeten die mutanten Remak-Schwannzellen unkompakte Membranwicklungen um nicht-myelinisierte C-Faser-Axone und um Kollagenfaserbündel aus („Remak-Myelin“). Sowohl in den Regionen unkompakten Myelins als auch in Remak- Schwannzellen konnte eine erhöhte Aktivität des PI3K-AKT-Signalwegs nachgewiesen werden. Vermutlich setzt die Anreicherung von PIP3 mit Überaktivierung des PI3K-AKT-Signalwegs in den mutanten Gliazellen einen zellautonomen Prozess der Umwicklung von Axonen in Gang. Die zusätzliche Bildung von „Remak-Myelin“ um Kollagenfasern, die keine Membranoberfläche besitzen, weist darauf hin, dass dieser Prozess nicht von einer bidirektionalen axo-glialen Kommunikation abzuhängen scheint. Die beobachteten Tomacula und Myelinausfaltungen zeigten Ähnlichkeiten mit Mausmodellen für hereditäre Neuropathien des Menschen, wie HNPP und CMT4B. Wir vermuten, dass PTEN im unkompakten Myelin unkontrolliertes Membranwachstum verhindert und dass eine gestörte Balance von Phosphoinositiden einen Pathomechanismus von tomaculösen Neuropathien darstellt. Somit identifizieren wir den PI3K-AKT-Signalweg als ein mögliches Ziel zukünftiger Therapiekonzepte für hereditäre Neuropathien des Menschen.

Indiana University-Purdue University Indianapolis (IUPUI) / Human traumatic spinal cord injuries (SCI) are primarily incomplete contusion or compression injuries at the cervical spinal level, causing immediate local tissue damage and a range of potential functional deficits. Secondary damage exacerbates initial mechanical trauma and contributes to function loss through delayed cell death mechanisms such as apoptosis and autophagy. As such, understanding the dynamics of cervical SCI and related intracellular signaling and death mechanisms is essential. Through behavior, Western blot, and histological analyses, alterations in phosphatase and tensin homolog (PTEN)/phosphatidylinositol-3-kinase (PI3K) signaling and the neuroprotective, functional, and mechanistic effects of administering the protein tyrosine phosphatase (PTP) inhibitor, potassium bisperoxo (picolinato) vanadium ([bpV[pic]) were analyzed following cervical spinal cord injury in rats. Furthermore, these studies investigated the combination of subacute Schwann cell transplantation with acute bpV(pic) treatment to identify any potential additive or synergistic benefits. Although spinal SC transplantation is well-studied, its use in combination with other therapies is necessary to complement its known protective and growth promoting characteristics. v The results showed 400 μg/kg/day bpV(pic) promoted significant tissue sparing, lesion reduction, and recovery of forelimb function post-SCI. To further clarify the mechanism of action of bpV(pic) on spinal neurons, we treated injured spinal neurons in vitro with 100 nM bpV(pic) and confirmed its neurprotection and action through inhibition of PTEN and promotion of PI3K/Akt/mammalian target of rapamycin (mTOR) signaling. Following bpV(pic) treatment and green fluorescent protein (GFP)-SC transplantation, similar results in neuroprotective benefits were observed. GFP-SCs alone exhibited less robust effects in this regard, but promoted significant ingrowth of axons, as well as vasculature, over 10 weeks post-transplantation. All treatments showed similar effects in forelimb function recovery, although the bpV and combination treatments were the only to show statistical significance over non-treated injury. In the following chapters, the research presented contributes further understanding of cellular responses following cervical hemi-contusion SCI, and the beneficial effects of bpV(pic) and SC transplantation therapies alone and in combination. In conclusion, this work provides a thorough overview of pathology and cell- and signal-specific mechanisms of survival and repair in a clinically relevant rodent SCI model.