Insect GDNF:TTC fusion protein improves delivery of GDNF to mouse CNS.

With a view toward improving delivery of exogenous glial cell line-derived neurotrophic factor (GDNF) to CNS motor neurons in vivo, we evaluated the bioavailability and pharmacological activity of a recombinant GDNF:tetanus toxin C-fragment fusion protein in mouse CNS. Following intramuscular injection, GDNF:TTC but not recombinant GDNF (rGDNF) produced strong GDNF immunostaining within ventral horn cells of the spinal cord. Intrathecal infusion of GDNF:TTC resulted in tissue concentrations of GDNF in lumbar spinal cord that were at least 150-fold higher than those in mice treated with rGDNF. While levels of immunoreactive choline acetyltransferase and GFRalpha-1 in lumbar cord were not altered significantly by intrathecal infusion of rGNDF, GDNF:TTC, or TTC, only rGDNF and GDNF:TTC caused significant weight loss following intracerebroventricular infusion. These studies indicate that insect cell-derived GDNF:TTC retains its bi-functional activity in mammalian CNS in vivo and improves delivery of GDNF to spinal cord following intramuscular- or intrathecal administration.

IGF-1:tetanus toxin fragment C fusion protein improves delivery of IGF-1 to spinal cord but fails to prolong survival of ALS mice.

To improve delivery of human insulin-like growth factor-1 (hIGF-1) to brain and spinal cord, we generated a soluble IGF-1:tetanus toxin fragment C fusion protein (IGF-1:TTC) as a secreted product from insect cells. IGF-1:TTC exhibited IGF-1 and TTC activity in vitro; it increased levels of immunoreactive phosphoAkt in treated MCF-7 cells and bound to immobilized ganglioside GT1b. In mice, the fusion protein underwent retrograde transport by spinal cord motor neurons following intramuscular injection, and exhibited both TTC- and IGF-1 activity in the CNS following intrathecal infusion. Analogous to the case with TTC, intrathecal infusion of the fusion protein resulted in substantial levels of IGF-1:TTC in spinal cord tissue extracts. Tissue concentrations of hIGF-1 in lumbar spinal cords of mice infused with IGF-1:TTC were estimated to be approximately 500-fold higher than those in mice treated with unmodified recombinant hIGF-1 (rhIGF-1). Like rhIGF-1, infusion of IGF-1:TTC reduced levels of IGF-1 receptor immunoreactivity in the same extracts. Despite raising levels of exogenous hIGF-1 in spinal cord, intramuscular- or intrathecal administration of IGF-1:TTC had no significant effect on disease progression or survival of high-expressing SOD1(G93A) transgenic mice. IGF-1:TTC may prove to be neuroprotective in other animal models of CNS disease or injury known to be responsive to unmodified IGF-1.

Recombinant GDNF: tetanus toxin fragment C fusion protein produced from insect cells.

Glial cell line-derived neurotrophic factor (GDNF) has potent survival-promoting effects on CNS motor neurons in experimental animals. Its therapeutic efficacy in humans, however, may have been limited by poor bioavailability to the brain and spinal cord. With a view toward improving delivery of GDNF to CNS motor neurons in vivo, we generated a recombinant fusion protein comprised of rat GDNF linked to the non-toxic, neuron-binding fragment of tetanus toxin. Recombinant GDNF:TTC produced from insect cells was a soluble homodimer like wild-type GDNF and was bi-functional with respect to GDNF and TTC activity. Like recombinant rat GDNF, the fusion protein increased levels of immunoreactive phosphoAkt in treated NB41A3-hGFRalpha-1 neuroblastoma cells. Like TTC, GDNF:TTC bound to immobilized ganglioside GT1b in vitro with high affinity and selectivity. These results support further testing of recombinant GDNF:TTC as a non-viral vector to improve delivery of GDNF to brain and spinal cord in vivo.

VEGF increases blood-brain barrier permeability to Evans blue dye and tetanus toxin fragment C but not adeno-associated virus in ALS mice.

Entry of most compounds into the CNS is impeded by the blood-brain barrier (BBB). Because vascular endothelial growth factor (VEGF) is important in the formation and maintenance of the BBB and is known to modulate BBB permeability in newborn rodents, we tested the hypothesis that VEGF may enhance BBB permeability in adult mice. We examined the effect of VEGF on the CNS distribution of three different agents: a small molecule (Evans blue dye) that is known to bind plasma proteins, an exogenous protein (tetanus toxin fragment C; TTC), and a viral vector (recombinant adeno-associated virus serotype 2/5 marked with lacZ; rAAV2/5-lacZ). Pretreatment with VEGF (20 mug; i.v.) increased permeability of the BBB to Evans blue dye and TTC as detected by augmented concentrations of these substances in the cerebrum, brainstem, and spinal cord. By contrast, VEGF did not alter BBB permeability to AAV2/5-lacZ, as defined by beta-galactosidase activity assay. These data demonstrate the potential utility of VEGF for pharmacological modulation of the BBB, and indicate that the increase in BBB permeability mediated by VEGF is limited by the size of the delivered substance.

A glial cell line-derived neurotrophic factor (GDNF):tetanus toxin fragment C protein conjugate improves delivery of GDNF to spinal cord motor neurons in mice.

Glial cell line-derived neurotrophic factor (GDNF) has shown robust neuroprotective and neuroreparative activities in various animal models of Parkinson's Disease or amyotrophic lateral sclerosis (ALS). The successful use of GDNF as a therapeutic in humans, however, appears to have been hindered by its poor bioavailability to target neurons in the central nervous system (CNS). To improve delivery of exogenous GDNF protein to CNS motor neurons, we employed chemical conjugation techniques to link recombinant human GDNF to the neuronal binding fragment of tetanus toxin (tetanus toxin fragment C, or TTC). The predominant species present in the purified conjugate sample, GDNF:TTC, had a molecular weight of approximately 80 kDa as determined by non-reducing SDS-PAGE. Like GDNF, addition of GDNF:TTC to culture media of neuroblastoma cells expressing GFRalpha-1/c-RET produced a dose-dependent increase in cellular phospho-c-RET levels. Treatment of cultured midbrain dopaminergic neurons with either GDNF or the conjugate similarly promoted both DA neuron survival and neurite outgrowth. However, in contrast to mice treated with GDNF by intramuscular injection, mice receiving GDNF:TTC revealed intense GDNF immunostaining associated with spinal cord motor neurons in fixed tissue sections. That GDNF:TTC provided neuroprotection of axotomized motor neurons in neonatal rats further revealed that the conjugate retained its GDNF activity in vivo. These results indicate that TTC can serve as a non-viral vehicle to substantially improve the delivery of functionally active growth factors to motor neurons in the mammalian CNS.

Tetanus toxin fragment C fusion facilitates protein delivery to CNS neurons from cerebrospinal fluid in mice.

To improve protein delivery to the CNS following intracerebroventricular administration, we compared the distribution of a human Cu/Zn superoxide dismutase:tetanus toxin fragment C fusion protein (SOD1:TTC) in mouse brain and spinal cord with that of tetanus toxin fragment C (TTC) or human SOD1 (hSOD1) alone, following continuous infusion into the lateral ventricle. Mice infused with TTC or SOD1:TTC showed intense anti-TTC or anti-hSOD1 labeling, respectively, throughout the CNS. In contrast, animals treated with hSOD1 revealed moderate staining in periventricular tissues. In spinal cord sections from animals infused with SOD1:TTC, the fusion protein was found in neuron nuclear antigen-positive (NeuN+) neurons and not glial fibrillary acidic protein-positive (GFAP+) astrocytes. The percentage of NeuN+ ventral horn cells that were co-labeled with hSOD1 antibody was greater in mice treated with SOD1:TTC (cervical cord = 73 +/- 8.5%; lumbar cord = 62 +/- 7.7%) than in mice treated with hSOD1 alone (cervical cord = 15 +/- 3.9%; lumbar cord = 27 +/-4.7%). Enzyme-linked immunosorbent assay for hSOD1 further demonstrated that SOD1:TTC-infused mice had higher levels of immunoreactive hSOD1 in CNS tissue extracts than hSOD1-infused mice. Following 24 h of drug washout, tissue extracts from SOD1:TTC-treated mice still contained substantial amounts of hSOD1, while extracts from hSOD1-treated mice lacked detectable hSOD1. Immunoprecipitation of SOD1:TTC from these extracts using anti-TTC antibody revealed that the recovered fusion protein was structurally intact and enzymatically active. These results indicate that TTC may serve as a useful prototype for development as a non-viral vehicle for improving delivery of therapeutic proteins to the CNS.

A survival motor neuron:tetanus toxin fragment C fusion protein for the targeted delivery of SMN protein to neurons.

Spinal muscular atrophy (SMA) is a degenerative disorder of spinal motor neurons caused by homozygous mutations in the survival motor neuron (SMN1) gene. Because increased tissue levels of human SMN protein (hSMN) in transgenic mice reduce the motor neuron loss caused by murine SMN knockout, we engineered a recombinant SMN fusion protein to deliver exogenous hSMN to the cytosolic compartment of motor neurons. The fusion protein, SDT, is comprised of hSMN linked to the catalytic and transmembrane domains of diphtheria toxin (DTx) followed by fragment C of tetanus toxin (TTC). Following overexpression in Escherichia coli, SDT possessed a subunit molecular weight of approximately 130 kDa as revealed by both SDS-PAGE and immunoblot analyses with anti-SMN, anti-DTx, and anti-TTC antibodies. Like wild-type SMN, purified SDT showed specific binding in vitro to an RG peptide derived from Ewing's sarcoma protein. The fusion protein also bound to cultured primary neurons in amounts similar to those achieved by TTC. Unlike the case with TTC, however, immunolabeling of SDT-treated neurons with anti-TTC and anti-SMN antibodies showed staining restricted to the cell surface. Results from cytotoxicity studies in which the DTx catalytic domain of SDT was used as a reporter protein for internalization and membrane translocation activity suggest that the SMN moiety of the fusion protein is interfering with one or both of these processes. While these studies indicate that SDT may not be useful for SMA therapy, the use of the TTC:DTx fusion construct to deliver other passenger proteins to the neuronal cytosol should not be ruled out.

The Ewing's sarcoma protein interacts with the Tudor domain of the survival motor neuron protein.

The survival motor neuron (SMN) gene is the spinal muscular atrophy (SMA) determining gene. Here we report that the SMN protein product interacts in vitro and in vivo with the arginine/glycine (RG)-rich RNA binding protein and transcription factor, Ewing's sarcoma (EWS). Recently, the SMN encoded Tudor domain (exon 3) and the YG-motifs (exon 6) have been shown to be involved in binding to RG-rich proteins. Here, we demonstrate that the Tudor domain encoded by SMN exon 3 is independently sufficient to mediate the interaction with EWS. Synthetic mutations within the Tudor domain, as well as a SMA patient-derived mutation within exon 3, reduced the levels of the SMN/EWS interaction. Carboxyl-terminal SMN mutations that prevent formation of SMN oligomers also indirectly reduced EWS binding. A role for arginine methylation has been observed in some RG-containing SMN-interacting proteins. Here we demonstrate that SMN interacts with non-methylated EWS and an EWS-derived RG-containing peptide. In contrast to previously reported results, symmetrical dimethylation of the EWS-derived RG-peptide results in a quantitative increase in the dissociation rate between SMN and the symmetrical dimethylated EWS RG-peptide. Consistent with the interaction data, endogenous and transiently expressed SMN co-localizes with endogenous EWS in a number of cultured cell lines, as well as rat primary neuron cultures. Anti-sense RNA experiments, however, demonstrate that EWS does not mediate the nuclear distribution of SMN or other Cajal body components.

Survival motor neuron protein in the nucleolus of mammalian neurons.

Spinal muscular atrophy (SMA) is an inherited motor neuron disease caused by mutations in the survival motor neuron gene (SMN1). While it has been shown that the SMN protein is involved in spliceosome biogenesis and pre-mRNA splicing, there is increasing evidence indicating that SMN may also perform important functions in the nucleolus. We demonstrate here through the use of a previously characterized polyclonal anti-SMN antibody, abSMN, that the SMN protein shows a striking colocalization with the nucleolar protein, fibrillarin, in both nucleoli and Cajal bodies/gems of primary neurons. Immunoblot analysis with antifibrillarin and two different anti-SMN antibodies reveals that SMN and fibrillarin also cofractionate in the insoluble protein fraction of cultured cell lysates. Immunoprecipitation experiments using whole cell extracts of HeLa cells and cultured neurons revealed that abSMN coprecipitated small amounts of the U3 small nucleolar RNA (snoRNA) previously shown to be associated with fibrillarin in vivo. These studies raise the possibility that SMN may serve a function in rRNA maturation/ribosome synthesis similar to its role in spliceosome biogenesis.