A Bicistronic Plasmid Encoding Brain-Derived Neurotrophic Factor and Urokinase Plasminogen Activator Stimulates Peripheral Nerve Regeneration After Injury

Abstract

Timely nerve restoration is an important factor for the successful regeneration of tissues and organs. It is known that axon regeneration following nerve injury is a multifactorial process that depends on the local expression of neurotrophins, including brain-derived neurotrophic factor (BDNF). Along with the survival of neurons, the active reorganization of the extracellular matrix is an important step for the growth of axons to their targets. Urokinase serine protease is part of the plasminogen activator system, which provides the vectoriality of the process of fibrinolysis and matrix reorganization, facilitating the growth of nerves to their targets. Based on this and in view of the results of our previous studies, we suggest that a combined bicistronic plasmid encoding the complementary proteins BDNF and urokinase may be beneficial in nerve regeneration. The ability of this bicistronic plasmid to stimulate nerve restoration was confirmed by in vitro stimulation of Neuro2a neurite growth and in vivo nerve conductivity and histology studies. To our knowledge, this is the first article that demonstrates the effectiveness of a bicistronic plasmid containing the human genes BDNF and urokinase plasminogen activator in the regeneration of the injured peripheral nerve. The results obtained demonstrate that plasmid vectors encoding several complementary-active therapeutic proteins may serve as a basis for developing prospective treatments for a wide range of multicomponent neural system disorders, such as nerve trauma. SIGNIFICANCE STATEMENTThis study is the first to show the effectiveness of using a bicistronic plasmid encoding complementary-active human protein brain-derived neurotrophic factor and urokinase plasminogen activator in the regeneration of the crushed peripheral nerve in a murine model.

Urokinase-Type Plasminogen Activator Enhances the Neuroprotective Activity of Brain-Derived Neurotrophic Factor in a Model of Intracerebral Hemorrhage

“…As a plasmid encoding hBDNF and huPA was effective for treating injured nerves [ 4 ], we considered it would stimulate brain recovery in the intracerebral hemorrhage model. However, we later abandoned this option because the production of a recombinant protein during gene therapy takes about 24–48 h, but a stroke, unlike nerve damage, is an emergent condition and requires an urgent intervention.…”

“…Conditioned medium samples were obtained from HEK293T cells transfected with pVax1, pVax1-hBDNF [ 10 ], pVax1-huPA, or pNCure-fIntr [ 4 ], denoted as Control, BDNF, uPA, and BU (BDNF + uPA), respectively. For this endeavor, transfected HEK293T cells were cultured in serum-free DMEM High Glucose for 48 h. Then medium samples were removed, centrifuged for 10 min at 3000× g to remove cell debris, and concentrated ~50 times using a Centriprep Centrifugal Filter Unit (Merck, #4302, Tullagreen, Ireland) until a hBDNF concentration of 3.5 ng/µL (108 nM) was reached, confirmed using the Human Free BDNF Quantikine ELISA Kit (R&D Systems, #DBD00, Minneapolis, MN, USA).…”

“…According to modern concepts, however, many proteins with different modalities (growth factors, cytokines, proteases, and matrix proteins) that compete with or complement each other are involved in the processes of development, functioning, and regeneration of tissues. Based on this fact, we showed that a combination of therapeutic genes with different modalities (BDNF and urokinase-type plasminogen activator (uPA)) is beneficial for peripheral nerve regeneration [ 4 ]. In our opinion, uPA is one of the most successful partners for BDNF for better stimulation of nervous tissue regeneration, because uPA is involved in a wide range of processes of nervous tissue ontogenesis and regeneration: navigation and outgrowth of neurites, fibrinolysis at the site of injury, and activation of growth factors, including BDNF [ 5 , 6 , 7 ].…”

“…Such outstanding pro-regenerative properties of BDNF and uPA could be applied for treating currently untreatable severe neurological conditions, such as intracerebral hemorrhage. A previously described bicistronic genetic construct, encoding human BDNF and uPA, efficiently stimulated restoration of the injured peroneal nerve [ 4 ]. Therefore, in the present study we explored the potential of a combination of human BDNF and uPA to stimulate brain tissue recovery after intracerebral hemorrhage.…”

New Frontiers in Peripheral Nerve Regeneration: Concerns and Remedies

“…Multiple studies indicate that the absence of endogenous BDNF impedes the growth of sciatic nerve axons and their myelination after injury [42,43], whereas local delivery of recombinant BDNF using a prolonged release capsule system significantly improves nerve regeneration [44]. We have recently developed a therapeutic approach using the local delivery of a genetic construct for a combined expression of BDNF and urokinase ensuring a long-term expression of these proteins at the site of nerve injury [45]. The abovementioned strategy is less stressful compared to serial protein injections.…”