Endogenous Mechanisms Of Neuroprotection: To Boost Or Not To Be Part 1

Abstract: 

Postmitotic cells, like neurons, must live through a lifetime. For this reason, organisms/cells have evolved with self-repair mechanisms that allow them to have a long life. 

Neurons are the basic units that make up the human brain. They transmit information through electrical signals and connect our thinking and memory with the outside world. Memory refers to the ability of humans to store and recall external information through perception, experience, and thinking, and neurons are an important part of this process.

As people age, the number and activity of neurons gradually decrease, which affects the level of memory. Therefore, we need to promote the health of neurons and memory by maintaining an optimistic attitude and healthy living habits.

First of all, maintaining an optimistic attitude is an effective way to improve neuronal activity. An optimistic attitude can stimulate the brain to release similar substances such as dopamine and catecholamines, thereby promoting neuronal activity and improving memory levels.

Secondly, maintaining healthy living habits is also essential for the health of neurons. Certain exercises and exercises can promote blood circulation and oxygen supply, which helps to improve the activity and memory level of neurons. In addition, good eating habits can also provide neurons with sufficient nutrition and promote memory improvement.

In short, the relationship between neurons and memory is inseparable. Maintaining an optimistic attitude and healthy living habits are effective ways to improve neuronal activity and memory levels. Let's maintain good living habits, keep an optimistic attitude, and build a happy, healthy, and energetic life together. It can be seen that we need to improve memory. Cistanche can significantly improve memory because it is a traditional Chinese medicinal material with many unique effects, one of which is to improve memory. The effect of Cistanche comes from the various active ingredients it contains, including tannic acid, polysaccharides, flavonoid glycosides, etc. These ingredients can promote brain health in many ways.

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The discovery workflow of neuroprotectors during the last years has focused on blocking the pathophysiological mechanisms that lead to neuronal loss in neurodegeneration. 

Unfortunately, only a few strategies from these studies were able to slow down or prevent neurodegeneration. There is compelling evidence demonstrating that endorsing the self-healing mechanisms that organisms/cells endogenously have, commonly referred to as cellular resilience, can arm neurons and promote their self-healing. 

Although enhancing these mechanisms has not yet received sufficient attention, these pathways open up new therapeutic avenues to prevent neuronal death and ameliorate neurodegeneration. Here, we highlight the main endogenous mechanisms of protection and describe their role in promoting neuron survival during neurodegeneration.

Keywords: autophagy; cellular resilience; endogenous mechanisms; neuroprotection; neuronal survival; unfolded protein response.

1. Neurodegenerative Processes

With increasing life expectancy in developed countries, the frequency of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD) or Huntington's disease (HD), or age-related decline of our nervous system performance, are likely to increase. 

Although there are several lines of evidence indicating that these pathologies have neuronal, astroglial, and microglial components, the decline in daily functions is caused by progressive neuronal loss. 

Due to their low turnover, neurons are postmitotic cells that must live for a lifetime. For this reason, they need powerful intrinsic protective machinery to cope with external and internal insults, which will cause their demise. 

These external/internal hazards are traumatic injuries or excitotoxic compounds, reactive oxygen species (ROS), protein aggregates, and other toxic molecules. 

Fortunately, cells have intrinsic machinery that blocks death by activating resilience mechanisms or promoting regeneration pathways. While young neurons have proper functioning of these self-healing protective mechanisms, aging disturbs them, leaving the neurons unprotected. In the same direction, dysfunctionality in these self-healing mechanisms has also been described in neurodegenerative diseases. 

During the last decades, enormous efforts have been invested in obtaining novel and effective neuroprotective therapies. 

However, they are intended to target pathophysiological mechanisms, which in the end turn into an acceleration of neuronal demise. Therefore, why not boost the mechanisms that neurons have naturally to obtain an effective neuroprotective approach?

This protective network is driven by the crosstalk of different cellular processes (i.e., unfolded protein response (UPR), autophagy, etc.), but they converge into the same process: allowing the cell to adapt to stress and survive [1–3]. 

Recently, we have considered a novel rationale to discover neuroprotectants: decipher what molecular mechanisms neurons engage after two different nerve injuries with opposite phenotypes, survival or death, which share similarities with health and neurodegeneration/aging. To do so, we used two in vivo-based peripheral nerve injury models that mimic the functionality or dysfunctionality of the endogenous mechanisms of protection. 

They provoke either motoneuron (MN) death (root avulsion (RA)) or survival (distal axotomy (DA)), depending on the soma–injury distances [2]. With the help of these models and using a Systems Biology-based approach, we confirmed that the death of MNs after RA shares similarities with the neuronal loss observed in neurodegenerative diseases, and we also described which mechanisms are used by MNs to survive after nerve injury [2]. 

Degenerative processes are apoptosis, necrosis, anoikis, endoplasmic reticulum (ER) stress, nucleolar stress, cytoskeletal rearrangements, and mitochondrial dysfunction, while the drivers of survival are: a correct UPR, the heat shock response, the autophagic pathway, the ubiquitin-proteasome system, the chaperone systems, the ER-associated degradation machinery and the antioxidant defense (Table 1). 

Interestingly, all these mechanisms have been separately described years ago and referred to as pre-conditioning injuries (see below).

We have demonstrated that boosting these endogenous mechanisms of neuroprotection through pharmacological treatment allows MN to survive in different pro-death scenarios, ranging from different species to different stages of development [23,54,55].

2. First Evidence of Endogenous Mechanisms: Pre-Conditioning

The phenotypic effects of endogenous mechanisms of protection were described 40 years ago. In 1986. Murry et al. described that sublethal physiological stress, also known as preconditioning injury, enhances tissue recovery in the heart [56]. From here, these healing mechanisms were also observed in the brain and spinal cord (SC) [57]. 

For example, these cellular responses are observed after nerve injury or during heart regeneration, where the production of ROS or extracellular vesicles respectively, drives functional recovery [58–60]. Surprisingly, the pre-conditioning of a specific organ exerts protection for others from injury [61]. Several specific effectors are responsible for these effects. 

After preconditioning injury, the production of different mediators (nitric oxide or ROS) will activate the signaling pathways phosphatidylinositol 3-kinase (PI3K)/Protein kinase B (AKT), Protein kinase C (PKC), and other signaling pathways that will modulate transcription factors such as Hypoxia-inducible factor 1-alpha (Hif1-α) or NF-κB. 

These will result in the production of nitric oxide synthases (iNOS), heat-shock proteins (HSPs), and cyclooxygenase-2 (COX-2), which are described as "end effectors", and will promote the protective effect within the tissue against future insults [61]. 

Together, these studies suggest that organisms/cells have endogenous protective mechanisms, and boosting them may be an effective therapeutic strategy.

3. Endogenous Mechanisms of Neuroprotection

3.1. Fine-Tuning Autophagy

Neurons require continuous recycling of intracellular materials to maintain homeostasis. Macro-autophagy, hereafter referred to as autophagy, is a highly coordinated molecular network in eukaryotic cells that pursues to recycle cytoplasmic content through lysosomal degradation. 

Although this degradation mechanism was initially observed only under starvation, recent studies showed that cells have a basal level of autophagy to regulate protein homeostasis. 

These basal levels are essential for axonal maintenance and survival of neurons under normal conditions [62,63]. A functional autophagic flux is a process highly coordinated by different autophagy-related (ATG) genes, kinases, and other regulatory proteins. They all work together to orchestrate the correct initiation, nucleation, elongation, closure, and fusion of autophagosomes with lysosomes to degrade the cytosolic load [64]. 

A reduced flow of autophagy is observed in the hippocampus during aging, while the reestablishment of its levels facilitates the formation of new memories [65]. 

Impaired or dysfunctional autophagy in neurons is associated with neurodegeneration, while activation of autophagy produces neuroprotection [5,54]. Alterations in the proteins related to the initial and elongation phases have been observed in amyotrophic lateral sclerosis (ALS) [66,67], and inducers of autophagy, such as rapamycin, exert neuroprotection after cerebral ischemia, traumatic brain injury (TBI), and AD [68–70]. 

The neuron-specific knockout (KO) of ATG5 or ATG7 causes neurodegeneration, accumulation of cytoplasmic inclusion bodies, and death of neurons [62,71], while their overexpression is beneficial in a model of PD [4]. 

Finally, p62, which manages the charge in the autophagosome and plays a key role in the late stages of autophagosome formation, is neuroprotective in fly models characterized by protein aggregates, which is a hallmark of neurodegenerative diseases [6]. 

Several studies have shown the accumulation of autophagosomes and autolysosomes during neurodegeneration, suggesting that autophagy is overactivated and may trigger neuronal death. 

Aberrant accumulation of autophagic processes within the cytoplasm may be caused by lysosomal dysfunction, rather than overactivated autophagy [72]. Autophagy is properly initiated after TBI, but autophagosomes are not eliminated due to lysosomal dysfunction, leading to unresolved autophagy that promotes neuronal death [73]. 

These non-functional lysosomal pathways are also seen after a spinal cord injury (SCI), hampering functional recovery [74]. A similar blockage in the clearance of autophagosomes is also described in neurodegenerative diseases (i.e., the human brains of AD) [75]. 

The integration of all this evidence suggests that enhancing the resolution of autophagy can yield protection. Platt recently highlighted the therapeutic avenue of improving the function of lysosomal proteins to prevent neurodegeneration [76]. 

The overexpression of transcription factor EB (TFEB), which modulates a transcriptional network essential for lysosome biogenesis and function, has promoted neuroprotective effects in a rat model of PD [7] and an AD mice model [8]. 

The induction of autophagy is not as good as we would like. Although it is a canonical protective mechanism, its machinery or overactivation can facilitate cell death [77,78]. 

Inhibition of autophagy after exposure to human prions reduces neuronal damage, indicating that induction of autophagy also drives death [79], and reduced autophagy initiation promotes functional recovery after SC hemisection, prevents apoptosis, and reduces pyramidal death after ischemia in neonatal and adult mice [80–82]. 

If we focus on axotomized neurons, blocking autophagy is neuroprotective for the rubrospinal ones [80], while an increase in the level of ATG5 protects spinal MNs [5]. Adding controversy, cancer cells treated with chemotherapy activate autophagy to overcome treatment-induced apoptotic death, while MN-dependent autophagy inhibits apoptosis [54]. 

Besides, ATGs also trigger neuronal death. ATG5 loses its pro-autophagic capabilities when cleaved, moving its activity toward the induction of cell death [83–85]. Beclin1 has anti-apoptotic effects under normal conditions, but its cleavage at the C-terminus sensitizes the cells to apoptotic signals [9]. 

Therefore, there is a crosstalk between both cellular processes, and the cells can redirect them to increase their chances of survival to cope with the insult [83]. So, what is important for neuroprotection? Boosting or blocking autophagy? 

Finetuning is the answer [86]. Induction of a fine-tuned autophagy yields beneficial effects by (i) removing non-functional proteins/organelles, (ii) allowing the cell to readapt to the new situation, and (iii) degrading harmful effects such as inflammation or apoptotic inducers [87,88], which mediate neuronal demise. 

However, this autophagy must be activated in a very specific window of time, avoiding excessive degradation that provokes cell death. 

Lastly, autophagy also has non-canonical/degradative functions, such as the modulation of the inflammatory response, the formation of new memories [65], the maintenance of synaptic homeostasis [89], and the transport of cargo within the cell [90]. So, complete blocking of it will lead to irreversible damage to the nervous system and/or neurons.

3.2. Tackling the Sexy Part of Unfolded Protein Response

Neurons are extremely sensitive to misfolded proteins and aggregates. 

The ER is responsible for cellular proteostasis, which is the synthesis, folding, and sorting of proteins. Any alteration in its fitness will lead to the accumulation of misfolded proteins, inducing ER stress and activating the ER-overload response (ERO), the ER-associated degradation (ERAD) pathways, or the UPR, which is a highly conserved cellular response. 

Alterations in the distribution and morphology of the ER and the UPR have been observed in neurodegenerative diseases [91–93] and when the neuron is isolated after a nerve injury [16,94]. 

Binding immunoglobulin protein (BIP), also known as GRP78, is an ER-resident chaperone that is the main sensor of the UPR. In the inactive state, BIP remains bound to the three major UPR effectors: the RNA-activated protein kinase-like ER kinase (PERK) which induces C/EBP homologous protein (CHOP), the inositol-requiring protein-1 alpha (IRE1α), which splices X-box binding protein 1 (Xbp1) mRNA, and the activating transcription factor-6 alpha (ATF6) [95,96]. 

When BIP detects misfolded proteins, these transducers are activated and drive changes in the gene expression of specific proteins (i.e., chaperones, transcription factors) to increase the cell's ability to correctly fold proteins by modulating gene expression, enhancing the clearance of misfolded proteins' clearance, or inhibiting protein synthesis, allowing the cell to adapt to the stress and survive [97]. 

As a proof of concept, BIP overexpression in dopamine neurons increases their survival, while its downregulation induces the death of nigral dopamine neurons [10]. Besides, BIP +/− mice show accelerated propagation of prion pathogenesis [98]. 

Overall, UPR modulation may exert protective effects on neurodegeneration [94], as reviewed recently by our group [99]. UPR activation is an early event in neurodegenerative diseases, and its precise modulation has beneficial effects on pathology progression [100,101]. Although UPR may act as an endogenous mechanism of cell protection, its (over)activation promotes apoptosis [102] (i.e., the PERK axis has pro- or anti-apoptotic capabilities [91]). 

Besides, recent evidence suggests that different perturbations of the ER will activate differentially the 3 branches of the UPR, indicating that the coordinated co-activation of them is not always present. Therefore, the cell has a specific program to respond to a specific insult. 

For instance, CHOP blockage or Xbp1 overexpression increases neuron survival after nerve injury, indicating that each branch has different roles in neuron death [16]. 

The early activation of PERK after brain injury exerts neuroprotection, while the sustained signaling through this pathway exacerbates cell loss [11]. Overexpression or pharmacological PERK activation reduces Tau pathology [12], while averting its sustained activation diminishes neuronal death [13] and improves age-related memory decline [14]. 

The inhibition of PERK in astrocytes delays neuronal loss in a prion-disease in vivo model. Interestingly, PERK activation in astrocytes disturbs the secretome, altering its synaptogenic function and causing synaptic loss [15]. 

The same authors described that the main downstream mechanisms involved in this detrimental effect of PERK are the extracellular matrix-cell adhesion pathways, which crosslink the UPR with the anoikis (see below, Section 3.4). 

Activating transcription factor 5 (ATF5) levels are directly dependent on the activation of PERK/eukaryotic translation initiation factor 2a (eIF2a). ATF5 has been directly linked to those neurons that are more resilient to death in human epilepsy [26]. 

However, the subsequent consequences of these effects are not as clear. ATF5 induces the expression of two anti-apoptotic effectors (see below), B-cell lymphoma 2 (Bcl-2) and induced myeloid leukemia cell differentiation protein (Mcl-1) [103], which will inhibit apoptosis. 

ATF5 also modulates the mechanistic target of rapamycin (mTOR) in non-neuronal tissues, which is the main modulator of autophagy, interrelating UPR and autophagy. 

Activation of IRE1α ameliorates liver failure [17], and its downstream effector Xpb1 promotes cardiac protection [18], and neuroprotection in AD, PD, and after stroke [19–21]. 

Strikingly, a study in diabetic and ischemia-induced retinopathy showed that the protective effects of UPR are mediated by Xbp1 [22]. Nonetheless, chronic activation of the IRE1α branch will lead to phosphorylation of the tumor necrosis factor-a (TNF-α) receptor-associated factor 2 (TRAF2), triggering apoptotic cell death in different ways [104–106]. 

Ectopic overexpression of Ire1α will lead to an autophagy-dependent neuronal death in a PD Drosophila model [107]. Therefore, an adjusted modulation of IRE1α-Xbp1 during a specific window may exert protection [108].

We recently described that NeuroHeal pharmacological treatment or sirtuin1 (SIRT1) overexpression induces survival of MN after nerve injury, and increases the presence of cleaved ATF6 while reducing IRE1α phosphorylation [23]. 

Pharmacological activation of ATF6 induces protection in different ischemia models by activating proteostasis [24], and the blockage of this transcription factor has deleterious effects. 

In detail, ATF6 modulates antioxidant-response-related proteins' expression, modulating the ROS hormesis [109]. Forced expression of ATF6 improves functional outcomes after stroke, and the authors suggest that this effect may be mediated by the induction of autophagy [25]. 

So, what is therapeutically interesting, activate, or attenuate UPR? The activation of specific branches of the UPR is the key point. Precise activation of the UPR can promote protective effects by helping the cell to restore proteostasis. 

Nonetheless, this concept should be taken with caution because if stress persists and proteostasis is not restored, the UPR triggers neuronal apoptosis which is mediated by the PERK or IRE1α branch [110]. In addition, UPR is also connected with autophagy and vice versa. 

BIP mediates the autophagic response, promoting neuronal survival [111]. Lastly, the 3 branches of UPR modulate the transcription of ATGs [112], suggesting an intricate link between both cellular processes.

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