Leveraging the ATP‐P2X7 receptor signalling axis to alleviate traumatic CNS damage and related complications

Yin, Yulong; Wei, Linyu; Caseley, Emily A.; Lopez‐Charcas, Osbaldo; Wei, Yingjuan; Li, Dongliang; Muench, Steve P.; Roger, Sebastian; Wang, Lu; Jiang, Lin‐Hua

doi:10.1002/med.21952

Cited by 6 publications

(3 citation statements)

References 201 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The 13A7 P2X7 receptor binding nanobody was chosen primarily for demonstration of proof-of-concept, but BBB penetrating P2X7 receptor binding agents have many potential therapeutic applications. Brain P2X7 receptor activation has been implicated in the pathophysiology of traumatic brain injury, post-traumatic headache, ischemic and hemorrhagic stroke, multiple sclerosis, neurodegenerative disorders, depression, and other conditions [ 48 – 58 ]. Development of P2X7 receptor binding reagents has been an active area of pharmaceutical research, and there remains an unmet need for highly specific, brain-penetrating P2X7 receptor antagonists [ 59 ].…”

Section: Discussionmentioning

confidence: 99%

Enhanced in vivo blood brain barrier transcytosis of macromolecular cargo using an engineered pH-sensitive mouse transferrin receptor binding nanobody

Esparza,

Su,

Francescutti

et al. 2023

Fluids Barriers CNS

View full text Add to dashboard Cite

Background The blood brain barrier limits entry of macromolecular diagnostic and therapeutic cargos. Blood brain barrier transcytosis via receptor mediated transport systems, such as the transferrin receptor, can be used to carry macromolecular cargos with variable efficiency. Transcytosis involves trafficking through acidified intracellular vesicles, but it is not known whether pH-dependent unbinding of transport shuttles can be used to improve blood brain barrier transport efficiency. Methods A mouse transferrin receptor binding nanobody, NIH-mTfR-M1, was engineered to confer greater unbinding at pH 5.5 vs 7.4 by introducing multiple histidine mutations. The histidine mutant nanobodies were coupled to neurotensin for in vivo functional blood brain barrier transcytosis testing via central neurotensin-mediated hypothermia in wild-type mice. Multi-nanobody constructs including the mutant M1R56H, P96H, Y102H and two copies of the P2X7 receptor-binding 13A7 nanobody were produced to test proof-of-concept macromolecular cargo transport in vivo using quantitatively verified capillary depleted brain lysates and in situ histology. Results The most effective histidine mutant, M1R56H, P96H, Y102H-neurotensin, caused > 8 °C hypothermia after 25 nmol/kg intravenous injection. Levels of the heterotrimeric construct M1R56H, P96H, Y102H-13A7-13A7 in capillary depleted brain lysates peaked at 1 h and were 60% retained at 8 h. A control construct with no brain targets was only 15% retained at 8 h. Addition of the albumin-binding Nb80 nanobody to make M1R56H, P96H, Y102H-13A7-13A7-Nb80 extended blood half-life from 21 min to 2.6 h. At 30–60 min, biotinylated M1R56H, P96H, Y102H-13A7-13A7-Nb80 was visualized in capillaries using in situ histochemistry, whereas at 2–16 h it was detected in diffuse hippocampal and cortical cellular structures. Levels of M1R56H, P96H, Y102H-13A7-13A7-Nb80 reached more than 3.5 percent injected dose/gram of brain tissue after 30 nmol/kg intravenous injection. However, higher injected concentrations did not result in higher brain levels, compatible with saturation and an apparent substrate inhibitory effect. Conclusion The pH-sensitive mouse transferrin receptor binding nanobody M1R56H, P96H, Y102H may be a useful tool for rapid and efficient modular transport of diagnostic and therapeutic macromolecular cargos across the blood brain barrier in mouse models. Additional development will be required to determine whether this nanobody-based shuttle system will be useful for imaging and fast-acting therapeutic applications.

show abstract

Section: Discussionmentioning

confidence: 99%

Enhanced in vivo blood brain barrier transcytosis of macromolecular cargo using an engineered pH-sensitive mouse transferrin receptor binding nanobody

Esparza,

Su,

Francescutti

et al. 2023

Fluids Barriers CNS

View full text Add to dashboard Cite

show abstract

“…The 13A7 P2X7 receptor binding nanobody was chosen primarily for demonstration of proof-ofconcept, but BBB penetrating P2X7 receptor binding agents have many potential therapeutic applications. Brain P2X7 receptor activation has been implicated in the pathophysiology of traumatic brain injury, posttraumatic headache, ischemic and hemorrhagic stroke, multiple sclerosis, neurodegenerative disorders, depression, and other conditions [48] [56] [57] [58]. Development of P2X7 receptor binding reagents has been an active area of pharmaceutical research, and there remains an unmet need for highly specific, brain-penetrating P2X7 receptor antagonists [59].…”

Section: Discussionmentioning

confidence: 99%

Enhancedin VivoBlood Brain Barrier Transcytosis of Macromolecular Cargo Using an Engineered pH-sensitive Mouse Transferrin Receptor Binding Nanobody

Esparza

Francescutti

et al. 2023

Preprint

View full text Add to dashboard Cite

Background: The blood brain barrier limits entry of macromolecular diagnostic and therapeutic cargos. Blood brain barrier transcytosis via receptor mediated transport systems, such as the transferrin receptor, can be used to carry macromolecular cargos with variable efficiency. Transcytosis involves trafficking through acidified intracellular vesicles, but it is not known whether pH-dependent unbinding of transport shuttles can be used to improve blood brain barrier transport efficiency. Methods: A mouse transferrin receptor binding nanobody, NIH-mTfR-M1, was engineered to confer greater unbinding at pH 5.5 vs 7.4 by introducing multiple histidine mutations. The histidine mutant nanobodies were coupled to neurotensin for in vivo functional blood brain barrier transcytosis testing via central neurotensin-mediated hypothermia in wild-type mice. Multi-nanobody constructs including the mutant M1R56H, P96H, Y102H and two copies of the P2X7 receptor-binding 13A7 nanobody were produced to test proof-of-concept macromolecular cargo transport in vivo using quantitatively verified capillary depleted brain lysates and in situ histology. Results: The most effective histidine mutant, M1R56H, P96H, Y102H -neurotensin, caused >8 degrees C hypothermia after 25 nmol/kg intravenous injection. Levels of the heterotrimeric construct M1R56H, P96H, Y102H -13A7-13A7 in capillary depleted brain lysates peaked at 1 hour and were 60% retained at 8 hours. A control construct with no brain targets was only 15% retained at 8 hours. Addition of the albumin-binding Nb80 nanobody to make M1R56H, P96H, Y102H -13A7-13A7-Nb80 extended blood half-life from 21 minutes to 2.6 hours. At 30-60 minutes, biotinylated M1R56H, P96H, Y102H -13A7-13A7-Nb80 was visualized in capillaries using in situ histochemistry, whereas at 2-16 hours it was detected in diffuse hippocampal and cortical cellular structures. Levels of M1R56H, P96H, Y102H-13A7-13A7-Nb80 reached more than 3.5 percent injected dose/gram of brain tissue after 30 nmol/kg intravenous injection. However, higher injected concentrations did not result in higher brain levels, compatible with saturation and an apparent substrate inhibitory effect. Conclusion: The pH-sensitive mouse transferrin receptor binding nanobody M1R56H, P96H, Y102H may be a useful tool for rapid and efficient modular transport of diagnostic and therapeutic macromolecular cargos across the blood brain barrier in mouse models. Additional development will be required to determine whether this nanobody-based shuttle system will be useful for imaging and fast-acting therapeutic applications.

show abstract

“…It has been proposed that ATP may act as a diffusible “danger signal” to alert about damage and start repair ( Dale, 2008 ). Because their rather low sensitivity ( Khakh and Alan North, 2006 ), P2X7r activation is a distinct feature of the injured CNS ( Cotrina and Nedergaard, 2009 ; Lecca et al, 2016 ; Territo and Zarrinmayeh, 2021 ; Yin et al, 2023 ). Using bioluminescence, Wang et al (2004) showed that ATP levels increase after SCI in a region spanning about 2 mm of tissue around the lesion epicenter.…”

Section: Discussionmentioning

confidence: 99%

P2X7 receptor activation awakes a dormant stem cell niche in the adult spinal cord

Falco,

Fabbiani,

Maciel

et al. 2023

Front. Cell. Neurosci.

View full text Add to dashboard Cite

The ependyma of the spinal cord is a latent stem cell niche that is reactivated by injury, generating new cells that migrate to the lesion site to limit the damage. The mechanisms by which ependymal cells are reactivated after injury remain poorly understood. ATP has been proposed to act as a diffusible “danger signal” to alert about damage and start repair. Indeed, spinal cord injury (SCI) generates an increase in extracellular ATP around the lesion epicenter that lasts for several hours and affects the functional outcome after the damage. The P2X7 receptor (P2X7r) has functional properties (e.g., low sensitivity for ATP, high permeability for Ca2+) that makes it a suitable candidate to act as a detector of tissue damage. Because ependymal cells express functional P2X7r that generate an inward current and regenerative Ca2+ waves, we hypothesize that the P2X7r has a main role in the mechanisms by which progenitor-like cells in the ependyma react to tissue damage. To test this possibility, we simulated the P2X7r activation that occurs after SCI by in vivo intraspinal injection of the selective agonist BzATP nearby the central canal. We found that BzATP rescued ependymal cells from quiescence by triggering a proliferative response similar to that generated by injury. In addition, P2X7r activation by BzATP induced a shift of ependymal cells to a glial fibrillary acidic protein (GFAP) phenotype similar to that induced by injury. However, P2X7r activation did not trigger the migration of ependyma-derived cells as occurs after tissue damage. Injection of BzATP induced the expression of connexin 26 (Cx26) in ependymal cells, an event needed for the proliferative reaction after injury. BzATP did not induce these changes in ependymal cells of P2X7–/– mice supporting a specific action on P2X7r. In vivo blockade of P2X7r with the potent antagonist AZ10606120 reduced significantly the injury-induced proliferation of ependymal cells. Our data indicate that P2X7r has a key role in the “awakening” of the ependymal stem cell niche after injury and suggest purinergic signaling is an interesting target to improve the contribution of endogenous progenitors to repair.

show abstract

Leveraging the ATP‐P2X7 receptor signalling axis to alleviate traumatic CNS damage and related complications

Cited by 6 publications

References 201 publications

Enhanced in vivo blood brain barrier transcytosis of macromolecular cargo using an engineered pH-sensitive mouse transferrin receptor binding nanobody

Enhanced in vivo blood brain barrier transcytosis of macromolecular cargo using an engineered pH-sensitive mouse transferrin receptor binding nanobody

Enhancedin VivoBlood Brain Barrier Transcytosis of Macromolecular Cargo Using an Engineered pH-sensitive Mouse Transferrin Receptor Binding Nanobody

P2X7 receptor activation awakes a dormant stem cell niche in the adult spinal cord

Contact Info

Product

Resources

About