Neurotrophins and the BBB Weihong Pan, M.D., Ph.D., Professor Blood-Brain Barrier Group Pennington Biomedical Research Center Baton Rouge, Louisiana, USA http://labs.pbrc.edu/bloodbrainbarrier Outline Overview of neurotrophins nomenclature, receptors, signaling pathways Neurotrophins in the CNS physiology and roles in neuroregeneration The blood-brain barrier (BBB) interacts with neurotrophins and neurotrophic cytokines General strategies to enhance CNS delivery of peptides and proteins Progress on neurotrophin delivery across the BBB Limitations and future directions 2 Neurotrophins and their receptors Member Nerve growth factor (NGF) p75ngfr + Specific receptor TrkA Brain-derived neurotrophic factor (BDNF) Neurotrophin-3 (NT-3) + + TrkB TrkC Neurotrophin-4/5 (NT-4/5) + TrkB 3 The screen versions of these slides have full details of copyright and acknowledgements 1
Neurotrophins Large proteins (118 120 aa) that form non-covalent homodimers (NGF even presents as a pentamer) Biological effects are determined by their specific interactions with the p75 NGFR and the ligand-selective tyrosine kinase receptors May stimulate growth of subpopulations of neurons as well as other cells Present in both the CNS and peripheral organs 4 Neurotrophin receptors and signals The principal domains in the Trks that determine specificity of binding are Ig-C2 p75 NGFR and Trks can all bind to neurotrophins with nanomolar affinity, though high affinity Trk binding sites in neuronal cells are present (Kd = 0.01 nm) The interactions and relative distributions of p75 NGFR and Trks affect biological effects Trk signaling involves phosphorylation of PLCγ, PI3K, and MAPK p75 NGFR belongs to the TNFR family and contains a cytoplasmic death domain; Signaling through ceramide, NFκB, and JNK may lead to cell death rather than survival 5 Other neurotrophic proteins Glial cell-derived neurotrophic factor Cilliary neurotrophic factor Hepatocyte growth factor S-100β Neuregulins Platelet-derived growth factor Vascular endothelial growth factor Pigment epithelium-derived growth factor Granulocyte/macrophage stimulatory factors Stem cell factors 6 The screen versions of these slides have full details of copyright and acknowledgements 2
Neurotrophic peptides (naturally occurring) Epidermal growth factor Transforming growth factors α and β Fibroblast growth factors a and b Pituitary adenylate cyclase activating peptide Insulin-like growth factor I Many other neurotrophic cytokines 7 Providing neurotrophic support to the brain and spinal cord Significance of the issue: therapeutic intervention to enhance cell survival and neurological functions The blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB): The rate-limiting factor The screen versions of these slides have full details of copyright and acknowledgements 3
The BBB: Ubiquitous 4-dimensional Protectiv e barrier Exchange interface Gate for drug deliv ery NGF 30 minutes after iv injection 11 The screen versions of these slides have full details of copyright and acknowledgements 4
Octanol/PBS partition coefficient 13 Capillary depletion 10 minutes after iv 14 Saturable transport of NGF Brain/serum ratio (µl/g) Saturable transport of NGF Time (min) 15 The screen versions of these slides have full details of copyright and acknowledgements 5
Saturable transport of NT3 16 BDNF 60 minutes after iv Gel electrophoresis of 125 I-BDNF after iv bolus injection, compared with the processing control and the 125 I-BDNF standard; There was no radioactivity at the bottom of the gel before run-off of the dye front Lane 1: brain 60 min after iv injection of 125 I-BDNF Lane 2: brain with addition of 125 I-BDNF ex vivo before homogenization Lane 3: serum 60 min after iv injection of 125 I-BDNF Lane 4: serum obtained by addition of 125 I-BDNF to collected blood Lane 6: 125 I-BDNF in PBS 17 BDNF entering brain parenchyma 18 The screen versions of these slides have full details of copyright and acknowledgements 6
Influx of BDNF by in-situ brain perfusion and effect of excess BDNF (2.5 µg/ml) Ki = 212.29 ± 26.74 µl/g-min Ki = 41.79 ± 18.03 µl/g-min 19 Brain radioactivity (cpm) Brain radioactivity (cpm) BDNF Albumin Time (min) log (cpm) = 0.007t + 4.134, r = 0.87, n = 8, P < 0.005 T½ = 46.4 min Excess BDNF (10 ng/µl) had no effect log (cpm) = 0.006t + 4.111, r = 0.92, n =8, P < 0.005 T½ = 52 min Classical neurotrophins: Are relatively stable in circulation Cross the BBB by saturable influx systems Vary in their permeability: BDNF > NGF > others Show regional differences in permeation: spinal cord > brain Display different degrees of reversible vascular association and parenchymal uptake Have no efflux transport 21 The screen versions of these slides have full details of copyright and acknowledgements 7
GDNF 30 minutes after iv administration 22 GDNF is relatively stable in circulation But does not show meaningful permeation across the BBB The screen versions of these slides have full details of copyright and acknowledgements 8
CNTF + excess (10 µg/mouse) 25 CNTF degradation assay 26 CNTF shows rather rapid degradation in vivo but may cross the BBB by a saturable transport system 27 The screen versions of these slides have full details of copyright and acknowledgements 9
LIF 28 LIF mainly shows reversible vascular binding 29 The influx is a saturable process 30 The screen versions of these slides have full details of copyright and acknowledgements 10
The spinal cord shows similar kinetics as in the brain 31 Saturation of influx is also seen in the blood-free in-situ brain perfusion system Saturation of influx is also seen in the blood-free in-situ brain perfusion system 32 A blocking antibody, but not CNTF, inhibits LIF influx 33 The screen versions of these slides have full details of copyright and acknowledgements 11
LIF: no efflux transport 34 Growth hormone: lack of saturable transport in mice and rats 40 125 I-GH Brain/Serum Ratio (µl/g) 30 20 10 c ontr ol with excess GH (1 µg /mou se ) 0 0 10 20 30 40 50 60 70 Exposure Time (m in ) 35 Growth hormone: no saturation shown by in-situ brain perfusion 10.0 125 I- GH only 125 I-GH Brain/Perfusate ( µl/g) 7.5 5.0 2.5 125 I-GH + unlabeled GH (20 µg/ml) 0.0 0 2 4 6 8 10 Time (min) 36 The screen versions of these slides have full details of copyright and acknowledgements 12
Growth hormone: significant uptake by brain parenchyma 37 4 0 Insulin and growth hormone do not compete at the BBB 125 I-Insulin Brain/Serum Ratio(µl/g) 3 0 2 0 1 0 1 2 5 I-in su li n, Ki = 0.9 7 ± 0.1 4 µl /g -mi n 1 2 5 I-in su li n+ G H, 10 µ g/mo us e, Ki = 0.7 1 ± 0.1 8 µ l/g -min 1 2 5 I-in su li n+ IG F 1, 2 µ g /mou se, K i = 0.32 ± 0.09 µ l/g -min 0 0 5 10 15 20 25 Exposure time (m in) 38 Insulin also does not interfere with IGF1 transport at physiological concentrations 39 The screen versions of these slides have full details of copyright and acknowledgements 13
In the absence of binding proteins, IGF-1 transport can be inhibited by insulin, shown by in-situ brain perfusion 40 EGF: example of a neurotrophic peptide Saturable influx 41 Intact 125 I-EGF can be recovered from brain after iv administration The screen versions of these slides have full details of copyright and acknowledgements 14
Most radioactivity in the whole brain represents EGF entering brain parenchyma 43 TGFα shares the transport system with EGF 44 A blocking antibody against EGFR showed no effect in reducing EGF influx 45 The screen versions of these slides have full details of copyright and acknowledgements 15
Mice without a functional EGF receptor maintain transport function 46 There is no efflux transport system for EGF 47 Enhancing CNS delivery of peptides and proteins Increase general permeab ilit y of the BBB Hyperosmo lar agents and bradykinin antagonists that target tight junctions Increase specific interactions of the molecule with the BBB Chemical modification to increase lipophilicit y Enhancing CNS delivery of peptides and proteins Increase positive charge for better adsorptive endocytosis Pegylatio n More stable analogs Smaller mimetics Conjugates targeting BBB-specific receptors Viral vectors Bypass the BBB Intranasal delivery Intracerebral/intrath ecal injections 48 The screen versions of these slides have full details of copyright and acknowledgements 16
Blood brain barrier targeting of BDNF improves motor function in rats with middle cerebral artery occlusion (Zhang & Pardridge, Brain Res. 2006) 49 Enhanced neuroprotective effects of basic fibroblast growth factor in regional brain ischemia after conjugation to a blood-brain barrier delivery vector Song et al., JPET 2002; Wu, NeuroRx 2005 bio-bfgf/ox26-sa conjugate 50 Application of a blood-brain-barrierpenetrating form of GDNF in a mouse model for Parkinson's disease (Dietz, Bahr, and colleagues, Brain Res. 2006) GDNF HIV-1-Tat-derived cell-penetrating peptide (CPP) MPTP-induced Parkinson s disease in mice Tat-GDNF reached DA neurons But no neuroprotection shown by the number of TH(+) neurons 51 The screen versions of these slides have full details of copyright and acknowledgements 17
Intranasal administration of insulin-like growth factor-i bypasses the blood brain barrier and protects against focal cerebral ischemic damage (Liu, Frey, and colleagues, J Neurol Sci 01) In 150 µg IGF-I significantly improved postural reflex and hemiparesis test 52 Left forepaw-placing test 53 Vestibulomotor function in the beam balance test 54 The screen versions of these slides have full details of copyright and acknowledgements 18
Brain-derived neurotrophic factor (BDNF) gene delivery into the CNS using bone marrow cells as vehicles in mice (Makar, Dhib-Jalbut and colleagues, Neurosci Lett 04) Detection of cell tracker orange labeled marrow cells in the striatum of an SJL mouse 10 weeks following tail vein injection Brain-derived neurotrophic factor (BDNF) gene delivery into the CNS using bone marrow cells as vehicles in mice (Makar, Dhib-Jalbut and colleagues, Neurosci Lett 04) 55 Mouse brain after transplantation with either BDNF provirus-transduced (Lanes 1, 4 and 5) or untransduced (Lanes 2, 3 and 6) bone marrow cells Mouse brain 3 months after bone marrow cell transplantion, showing GAD67 mrna and β-actin Limitations Paucity of BBB-specific markers for delivery Incomplete information on the trafficking and transcytosis processes Side-effects related to the carrier Limitations Dynamic changes of the BBB Many other factors in addition to neurotrophic support 57 The screen versions of these slides have full details of copyright and acknowledgements 19
Tsai & Tator CPD 2005 Summary Selective neurotrophins and neurotrophic peptides cross the BBB The passage may be mediated by simple diffusion or saturable transport system In CNS pathology with greater demand for neurotrophic support, different delivery strategies show variable success The BBB confers limitations and also advantages 59 References Pan W, Banks WA, Kastin AJ. Brain Res. 1998; 788: 87-94 Pan W, Banks WA, Fasold M, Bluth J, Kastin AJ. Neuropharm. 1998; 37: 1553-61 Pan W, Kastin AJ. Peptides 1999; 20: 1091-8 Pan W, Kastin AJ, Brennan JM. J Neuroimmunol. 2000; 106: 172-80 Pan W, Kastin AJ. Adv Drug Deliv Rev. 1999;36:291-298 Pan W, Kastin AJ. Neuroendocrinol. 2000; 72: 171-8 Liu XF et al., J Neurol Sci. 2001;187: 91-7 Song BW et al., 2002; 301: 605-10 Pan W, Akerstrom V, Kastin AJ. Neurosci Lett. 2003; 340: 239-41 Makar TK, Neurosci Lett. 2004; 356: 215-9 Pan W, et al., Endocrinol. 2005; 146: 4898-904 Tsai EC, Tator CH. Curr Pharm Des. 2005; 11: 1211-22 Wu D, NeuroRx. 2005; 2: 120-8 Yu Y, Kastin AJ, Pan W. Endocrinol. 2006; 147: 2611-5 Zhang Y, Pardridge WM, Brain Res. 2006; 1111: 227-9 Dietz GP 2006; 1082:61-6 60 The screen versions of these slides have full details of copyright and acknowledgements 20
61 The screen versions of these slides have full details of copyright and acknowledgements 21