Excitotoxicity-induced Endocytosis As A Potential Target For Stroke Neuroprotection Part 2

Prevention of TrkB-FL endocytosis is relevant for stroke neuroprotection in vivo: Since endocytosis is a requirement for necrosis, general strategies to downregulate or deplete key proteins mediating different steps of the endocytic process have been devised and shown to interfere with neurodegeneration (Troulinaki and Tavernarakis, 2012). 

In recent years, stroke has become a common problem, and because the neurological damage caused by a stroke will directly affect our daily lives, memory loss is relatively common after a stroke. Therefore, the relationship between neuroprotection and memory after stroke has also become a hot topic.

First of all, we need to understand that after a stroke, the nerves in the brain will be damaged to a certain extent, which will have a direct impact on our memory. However, if we take timely treatment and preventive measures, we can still effectively protect our nerves and improve our memory.

How to perform neuroprotection? First, we need to do proper exercise. Not only does this improve our physical condition, but it also promotes nerve growth and recovery. Secondly, developing good living habits, such as maintaining adequate sleep and eating properly, will also be of great help to our neuroprotection. Finally, during the treatment period, we need to actively cooperate with the doctor's treatment and receive rehabilitation training promptly, to effectively restore our neurological function.

In addition, we can also improve our memory through some methods. For example, we can develop good habits such as reading, listening to music, and playing intellectual games, which can stimulate our brains and promote the formation and maintenance of memories. At the same time, we can also try some memory training, such as reciting words, numbers, geographical locations, etc. These methods can effectively improve our memory ability.

Although stroke will bring a lot of inconvenience to our lives, as long as we actively cooperate with treatment and rehabilitation training, and develop good living and study habits, we can still live an active and healthy life and protect our nerves and memory. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory, because Cistanche deserticola has antioxidant, anti-inflammatory, and anti-aging effects, which can help reduce oxidation and inflammatory reactions in the brain, thereby protecting the health of the nervous system. In addition, Cistanche deserticola can also promote the growth and repair of nerve cells, thus enhancing the connectivity and function of neural networks. These effects can help improve memory, learning, and thinking speed, and may also prevent the development of cognitive dysfunction and neurodegenerative diseases.

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Therefore, generic i nte r fe re n c e o f e n d o c y to s i s m i g ht b e is considered a therapeutic strategy with the potential to reduce cell death due to acute brain insults. However, potential caveats to this approach are the pleiotropic adverse effects it might have considering that essential physiological cell processes would be inhibited as well. As an alternative, we propose the use of molecules able to interfere with pathological endocytosis of specific neuronal survival proteins.
One candidate to consider is the NMDAR, internalized by its overstimulation. Endocytosis of GluN2B-containing NMDARs has been shown to mediate excitotoxicity by still unknown mechanisms (Wu et al., 2017).
Blockade of this clathrin-dependent endocytic process using a cell-penetrating peptide (CPP) was able to inhibit excitotoxicity in cultured cortical neurons. This CPP (Tat-YEKL) contains an 11 aa domain of the human immunodeficiency virus type 1 Tat protein, which allows attached cargoes to cross the blood-brain barrier and plasma membrane, followed by 12 aa of the GluN2B C-terminus corresponding to the AP-2 binding motif. 

The selection of CPPs as neuroprotective tools for stroke therapy seems particularly appropriate NEURAL REGENERATION RESEARCH｜Vol 16｜No.2｜February 2021｜301 Figure 1 ｜ Mechanism of action of neuroprotective peptide TFL457. The CPP contains a TrkB-FL juxtamembrane sequence (dark green rectangle) that probably competes with a TrkB-interacting protein (orange) important for excitotoxicity-induced endocytosis, thus preventing secondary receptor processing and neuronal death. 

The interaction of TrkB with this unidentified protein is maintained in the presence of the control peptide TMyc (purple circle) and neuronal death is induced by the excitotoxic insult. CPP: Cell-penetrating peptide; TFL457 and TMyc: Tat-derived CPPs; TrkB: tropomyosin-related kinase B; TrkB-FL: the catalytically active full-length receptor. 

since the highly endocytic neurons of the ischemic area present an enhanced uptake of Tat-derived peptides (Vaslin et al., 2009) which results in selective targeting of potential neuroprotective CPPs to damaged neurons. In the future, it will be interesting to establish if this CPP has similar neuroprotective effects when used in vivo and can specifically inhibit the pro-death effects of NMDAR overactivation while preserving other receptor functions. 

Another possible candidate to be explored as the neuroprotective target is the kinase D-interacting substrate of 220 kDa (Kidins220), a downstream effector of neurotrophin receptors and NMDARs essential for neuronal viability. We have demonstrated that Kidins220 processing by calpain in excitotoxicity is secondary to the traffic of this protein to the Golgi apparatus and early activation of Rap1-GTPase (LópezMenéndez et al., 2019). 

At later stages of the excitotoxic process, Kidins220 downregulation governs Rap1 inactivation associated with a decrease in ERK activity which compromises neuronal survival. Therefore, prevention of excitotoxicity-induced Kidins220 endocytosis might be investigated as a way to inhibit calpain processing of Rap1 activation complexes and, thus, interfere with the shut-off of Kidins220/Rap1/ ERK prosurvival cascades. 

Finally, we have already demonstrated that interference of TrkB-FL endocytosis is a relevant neuroprotective strategy both in vitro and in vivo. We have found that excitotoxicity decreases the surface levels of this receptor and, sequentially, promotes its intracellular processing by several proteases (Tejeda et al., 2019). The major mechanism of TrkB-FL downregulation is the cleavage of its intracellular juxtamembrane region by calpain (Vidaurre et al., 2012; Figure 1). 

A secondary mechanism for TrkB-FL, but primary for TrkB-T1 regulation, is regulated intramembrane proteolysis by sequential metalloproteinase/γ-secretase action, producing the shedding by these isoforms of identical receptor ectodomains that we have demonstrated act as BDNFscavengers (Tejeda et al., 2016). 

Altogether, these mechanisms cause a profound alteration of BDNF signaling by excitotoxicity, observed not only in stroke but also in other neurological disorders (Tejeda and Diaz-Guerra, 2017). 

To preserve BDNF-regulated survival pathways, we have designed a Tat-derived CPP (TFL 457) containing a short TrkB-FL juxtamembrane sequence (aa 457–471) that we hypothesized might be important for the control of receptor stability and function in excitotoxicity. 

The selected sequence is part of an intracellular region established as important for the regulation of TrkB-FL location and function through interaction with a set of different proteins that include Hrs (Huang et al., 2009). 

We observed that peptide TFL457 specifically prevented early TrkB-FL endocytosis activated by excitotoxicity, differently from a negative control CPP containing non-related sequences (TMyc). We hypothesized that TFL457 might be competing with some protein/s interaction/s established by sequence 457–471 in TrkB-FL which would be required for the promotion of excitotoxicity-induced endocytosis. 

Since this peptide will only affect interactions specifically established by TrkB-FL, endocytosis of other surface proteins would not be affected. By keeping the unprocessed receptor in the cell surface, TFL457 secondarily interferes with TrkB-FL cleavage by calpain and regulated intramembrane proteolysis and, thus, receptor Tyr816 phosphorylation and BDNF/TrkB/ PLCγ-signaling are maintained. 

The result is the preservation of cAMP response-element binding protein and myocyte enhancer factor 2 promoter activities downstream this cascade, which starts a feedback mechanism favoring increased expression of critical prosurvival proteins in neurons, such as BDNF or the TrkB receptor itself, resulting in increased neuronal viability. 

The developed neuroprotective peptide could be highly relevant for stroke therapy since, in a mouse model of permanent ischemia, it counteracts TrkB-FL downregulation in the infarct and efficiently decreases infarct size by nearly 30% (Tejeda et al., 2019). 

Furthermore, TFL457 improves the neurological outcome, as shown by a decrease of 42% in the average number of slips in a test evaluating balance and motor coordination. 

Therefore, these results unravel endocytosis of TrkB-FL induced by excitotoxicity as a novel and highly relevant target for stroke therapy and open up new avenues for the development of novel drugs specifically directed to other survival proteins.

Margarita Díaz-Guerra*

Instituto de Investigaciones Biomédicas "Alberto Sols", Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), Madrid, Spain

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Open peer reviewer: Rayudu Gopalakrishna, University of Southern California, USA. Additional file: Open peer review report 1.

References

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2. Huang SH, Zhao L, Sun ZP, Li XZ, Geng Z, Zhang KD, Chao MV, Chen ZY (2009) Essential role of Hrs in endocytic recycling of full-length TrkB receptor but not its isoform TrkB.T1. J Biol Chem 284:15126-15136.

3. López-Menéndez C, Simón-García A, Gamir-Morralla A, Pose-Utrilla J, Luján R, Mochizuki N, Díaz-Guerra M, Iglesias T (2019) Excitotoxic targeting of Kidins220 to the Golgi apparatus precedes calpain cleavage of Rap1- activation complexes. Cell Death Dis 10:535.

4. Rudinskiy N, Grishchuk Y, Vaslin A, Puyal J, Delacourte A, Hirling H, Clarke PG, Luthi-Carter R (2009) Calpain hydrolysis of alpha- and beta2-adaptins decreases clathrin-dependent endocytosis and may promote neurodegeneration. J Biol Chem 284:12447-12458.

5. Scott DB, Michailidis I, Mu Y, Logothetis D, Ehlers MD (2004) Endocytosis and degradative sorting of NMDA receptors by conserved membrane-proximal signals. J Neurosci 24:7096-7109.

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