Kris Poulsen

Persephin, a Novel Neurotrophic Factor Related to GDNF and Neurturin

A novel neurotrophic factor named Persephin that is approximately 40% identical to glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) has been identified using degenerate PCR. Persephin, like GDNF and NTN, promotes the survival of ventral midbrain dopaminergic neurons in culture and prevents their degeneration after 6-hydroxydopamine treatment in vivo. Persephin also supports the survival of motor neurons in culture and in vivo after sciatic nerve axotomy and, like GDNF, promotes ureteric bud branching. However, in contrast to GDNF and NTN, persephin does not support any of the peripheral neurons that were examined. Fibroblasts transfected with Ret and one of the coreceptors GFRalpha-1 or GFRalpha-2 do not respond to persephin, suggesting that persephin utilizes additional, or different, receptor components than GDNF and NTN.

A GPI-linked protein that interacts with Ret to form a candidate neurturin receptor.

Glial-cell-line-derived neurotrophic factor (GDNF) and neurturin (NTN) are two structurally related, potent survival factors for sympathetic, sensory and central nervous system neurons. GDNF mediates its actions through a multicomponent receptor system composed of a ligand-binding glycosyl-phosphatidylinositol (GPI)-linked protein (designated GDNFR-α) and the transmembrane protein tyrosine kinase Ret. In contrast, the mechanism by which the NTN signal is transmitted is not well understood. Here we describe the identification and tissue distribution of a GPI-linked protein (designated NTNR-α) that is structurally related to GDNFR-α. We further demonstrate that NTNR-α binds NTN (Kd ∼ 10 pM) but not GDNF with high affinity; that GDNFR-α binds to GDNF but not NTN with high affinity; and that cellular responses to NTN require the presence of NTNR-α. Finally, we show that NTN, in the presence of NTNR-α, induces tyrosine-phosphorylation of Ret, and that NTN, NTNR-α and Ret form a physical complex on the cell surface. These findings identify Ret and NTNR-α as signalling and ligand-binding components, respectively, of a receptor for NTN and define a novel family of receptors for neurotrophic and differentiation factors composed of a shared transmembrane protein tyrosine kinase and a ligand-specific GPI-linked protein.

TGFβ2 and TGFβ3 are potent survival factors for midbrain dopaminergic neurons

The vertebrate ventral midbrain contains 3-4 x 10(4) dopaminergic neurons that influence motor activity, emotional behavior, and cognition. Recently, glial cell line-derived neurotrophic factor (GDNF) was shown to be a potent survival factor for these dopaminergic neurons in culture. However, many midbrain dopaminergic neurons project to targets that do not express GDNF. We report here that transforming growth factors (TGFs) TGF beta 2 and TGF beta 3, which are distantly related to GDNF, also prevent the death of cultured rat embryonic midbrain dopaminergic neurons at picomolar concentrations. Furthermore, we find that TGF beta 2, TGF beta 3, and GDNF are expressed sequentially as local and target-derived trophic factors and that subpopulations of dopaminergic neurons projecting to distinct targets have access to only one of these factors. These findings are consistent with the idea that GDNF, TGF beta 2, and TGF beta 3 are physiological survival factors for developing midbrain dopaminergic neurons and may have applications as therapeutics for Parkinsons disease, a neurodegenerative disorder of dopaminergic neurons.

Control of neuronal diversity by the floor plate: Contact-mediated induction of midbrain dopaminergic neurons

The notochord and floor plate contribute to patterning the ventral neural tube in part by expressing a diffusible factor that induces motoneurons. To determine the mechanisms that direct the development of other classes of ventral neurons, we studied the development of dopaminergic neurons that reside near motoneurons in the ventral midbrain. We provide evidence that dopaminergic neurons develop in the vicinity of the floor plate and that they can be specified by the floor plate in vitro and in vivo. Unlike motoneurons, efficient induction of dopaminergic neurons requires contact with floor plate cells. These results suggest that neuronal diversification along the dorsal-ventral axis may be achieved partly through the concerted action of diffusible and contact-dependent signals from a single organizing center, the floor plate.

Effect of attachment site on stability of cleavable antibody drug conjugates.

The systemic stability of the antibody-drug linker is crucial for delivery of an intact antibody-drug conjugate (ADC) to target-expressing tumors. Linkers stable in circulation but readily processed in the target cell are necessary for both safety and potency of the delivered conjugate. Here, we report a range of stabilities for an auristatin-based payload site-specifically attached through a cleavable valine-citrulline-p-aminobenzylcarbamate (VC-PABC) linker across various sites on an antibody. We demonstrate that the conjugation site plays an important role in determining VC-PABC linker stability in mouse plasma, and that the stability of the linker positively correlates with ADC cytotoxic potency both in vitro and in vivo. Furthermore, we show that the VC-PABC cleavage in mouse plasma is not mediated by Cathepsin B, the protease thought to be primarily responsible for linker processing in the lysosomal degradation pathway. Although the VC-PABC cleavage is not detected in primate plasma in vitro, linker stabilization in the mouse is an essential prerequisite for designing successful efficacy and safety studies in rodents during preclinical stages of ADC programs. The divergence of linker metabolism in mouse plasma and its intracellular cleavage offers an opportunity for linker optimization in the circulation without compromising its efficient payload release in the target cell.

Site-specific conjugation improves therapeutic index of antibody drug conjugates with high drug loading

Site-specific conjugation improves therapeutic index of antibody drug conjugates with high drug loading

Correction: A GPI-linked protein that interacts with Retto form a candidate neurturin receptor

This corrects the article DOI: 10.1038/42722