Chaperone-Mediated Autophagy in Neurodegenerative Diseases And Acute Neurological Insults in The Central Nervous System Part 3

6. Acute Neurological Insults and CMA

6.1. Traumatic Brain Injury

TBI initiates a cascade of multiple pathophysiological processes, including the degradation pathway of aberrant proteins, such as macroautophagy and UPS [96,97]. Thesedegradation systems are considered to be activated in response to various stress conditionsafter TBI. Decreasing toxic aberrant proteins via the autophagic process may provide aneuroprotective effect following TBI [98,99]. 

Importantly, a previous study showed thatLAMP2A expression increased in neurons and proliferated microglia in a rat model ofTBI [16]. In that study, the upregulation of LAMP2A occurred from 3 to 15 days followingTBI. 

Another study using a mouse model of TBI also demonstrated that LAMP2A expression was upregulated in the injured brain [100]. These findings suggested that the CMApathway can be activated in damaged neural tissue after TBI. 

A recent study demonstrated that annexin A1 peptide Ac2-26 activated the CMAprocess to degrade IKK and consequently reduced TNF- expression in microglial cultures [101]. 

These findings suggest that there is an anti-inflammatory mechanism associatedwith the CMA process in microglia [101]. Interestingly, silent information regulator 1 (Sirt1)activated CMA by upregulating DnaJ heat-shock protein family member B1 (Dnajb1) expression and consequently attenuated astrocyte activation and neuronal loss after TBI inmice [100]. 

Taken together, these findings suggest that the activation of the CMA pathwayfollowing TBI might exert a neuroprotective effect of attenuating inflammatory reactionsand reducing neural tissue damage in the injured brain [100]. 

6.2. Cerebral Ischemia

Ischemic cerebral stroke is one of the leading causes of death and morbidity in humans.Previous studies have suggested that over-activation of autophagic pathways exerts aneuroprotective effect in ischemic brain injury [102,103]. 

Hsc70 and Hsp40 are reported tobe synergistically expressed in the neurons of vulnerable areas in response to sub-lethalischemia [104]. The combination of Hsc70 and Hsp40 suppresses aggregate formation andapoptosis in neurons [105].

Another study showed that the upregulation of LAMP-2Aexpression and the accumulation of LAMP-2A-positive lysosomes were induced underischemic conditions in neuronal cells in vitro [17]. In an animal model of cerebral ischemia,LAMP-2A expression was slightly decreased until two days after ischemia and then the levelincreased significantly seven days after ischemia [17]. 

These findings suggest that CMA maybe activated under ischemic conditions in the brain and may facilitate neuronal survival.Blocking LAMP-2A expression with siRNA increased neuronal cell death after brainischemia. [17]. 

In addition, the administration of mycophenolic acid, a potent CMA activator, rescued hypoxia-mediated cell death in a brain ischemia model. Furthermore, amembrane-permeable peptide that specifically binds to cyclin-dependent kinase 5 (CDK5)with a CMA targeting motif (Tat-CDK5-CTM) can promote the degradation of CDK5, reducing neuronal cell death [106]. 

In addition, Tat-CDK5-CTM also reduced the infarctionarea and neuronal loss and improved the neurological functions in a cerebral infarctionmouse model [106]. Taken together, these findings suggest that promoting CMA activitymay lead to the acceleration of the removal of damaged protein, thereby contributing tothe survival of neurons after cerebral ischemia.

6.3. Spinal Cord Injury

Degradation of dysfunctional intracellular components via the autophagic process isa crucial step in maintaining cellular homeostasis in response to various forms of stress,including nutrient deprivation, hypoxia, reactive oxygen species, DNA damage, and endoplasmic reticulum (ER) stress [15,99,107,108]. 

Many previous studies have providedexperimental evidence that autophagy is an essential cytoprotective pathway for reducingsecondary neural tissue damage and functional impairment after SCI [99,109–111]. 

Wepreviously reported that LAMP2A protein expression was significantly upregulated indamaged neural tissue after SCI in mice [18]. The expression of LAMP2A was increasedin various neural cells, such as neurons, astrocytes, oligodendrocytes, and microglia, inthe injured spinal cord [18]. These results indicated that CMA was activated in damagedneural tissue following SCI. 

Interestingly, our results also showed that the number ofLAMP2A-expressing cells increased from 24 h and peaked at 3 days, lasting for at least7 days after injury. The time course of LAMP2A expression is similar to that of apoptosisafter SCI [112–114]. 

Apoptosis is considered a major cause of secondary damage followingSCI [112,114]. Therefore, CMA activity might be regulated in response to secondary neuraltissue damage. 

A previous study showed that histone deacetylase-6 (HDAC6) has a molecular function of inducing Hsp90 deacetylation and increasing the interaction between LAMP2A andHsp90, thereby upregulating CMA activity [115]. 

Another study showed that a deficiencyin HDAC6 hindered CMA activity to resist oxidative stress in vitro [116]. In addition,inhibition of HDAC6 accelerated reactive oxygen species (ROS) generation and neuronalapoptosis in response to hypoxia-ischemia [116]. 

Importantly, both HDAC6 and LAMP2Aexpressions are upregulated in a mouse model of SCI [116]. Takentogether, HDAC6 may have an important role in the regulation of CMA activity and maybe a potential therapeutic target for the effective treatment of SCI. 

Further studies will beneeded to clarify the pathophysiological and cytoprotective mechanisms of CMA after SCI.In summary, previous studies have shown evidence that CMA activity can be upregulated in damaged neural tissue following various types of acute neurological insults,such as cerebral infarction [17], TBI [16], and SCI [18]. 

7. Therapeutic Potential of CMA for Neurodegenerative Diseases

Major neurodegenerative diseases are generally caused by the accumulation of aberrant proteins, as described above. Aberrant proteins, such as -synuclein and LRRK2 inPD, RCAN1 and Tau protein in AD, Htt in HD, and TDP-43 in ALS and FTLD, are thesubstrates of CMA [4,14]. Thus, the upregulation of CMA activity has therapeutic potentialfor treating neurodegenerative diseases caused by misfolded proteins [15]. 

As a therapeuticapproach, CMA activity can be modulated by various molecular mechanisms, such aschanging the LAMP2A level in lysosomes, changing the Hsc70 level, and changing thecondition of the KFERQ-like motif.Many studies have suggested that enhancing LAMP2A expression to upregulatethe activity of CMA can be an important therapeutic target. 

A previous study demonstrated that recombinant adeno-associated virus augmenting the LAMP2A level protected dopaminergic neurons in the substantia nigra from -synuclein-induced degeneration [117]. 

In addition, it has also been reported that various compounds, such asgeldanamycin [118], 6-aminonicotinamide [119], glucose-6-phosphate dehydrogenase inhibitor [119], silymarin [120], chronic caffeine [121], manganese [122], trehalose [123],b-asarone [124], and other compounds extracted from natural medicinal plants [125],or even combination treatments with bortezomib and suberoylanilide hydroxamic acid(SAHA) [126], can increase LAMP2A levels and activate the CMA pathway. 

It was also reported that histone deacetylase 10 (HDAC10) deacetylates Hsc70 and upregulates the CMA pathway in vitro [127]. In addition, HDAC10 knock-out in cellsresults in the accumulation of LAMP2A-positive lysosomes around the nucleus, activatingCMA to degrade a well-known CMA substrate, GAPDH [128]. These findings suggesttherapeutic potential in the regulation of Hsc chaperones for CMA activation. 

Another therapeutic approach involves the modification of the condition of the KFERQ-like motif of pathological proteins to make them suitable for degradation via the CMApathway. A recent study showed that tagging amyloid- oligomers with multiple KFERQmotifs promoted their entering endosomes and lysosomes, thereby protecting humanprimary cultured cortical neurons from neurotoxicity [82]. 

In addition, the use of anadaptor containing two copies of polyQ binding sequences and two different KFERQmotifs specifically directed mutant Htt to CMA degradation, ameliorating symptoms in anHD disease model [62]. An artificial peptide containing two CMA recognition motifs fusedto two copies of the polyglutamine-binding peptide 1 (QPB1) sequence enables Htt to bedegraded by CMA, ameliorating Htt aggregation and toxicity [62]. 

Interestingly, a novelantibody containing a KFERQ-like motif was able to recognize TDP-43 and targeted itto lysosomes for CMA degradation [129]. These findings suggest that modification of thecondition of the KFERQ-like motif of aberrant proteins may be a new therapeutic strategyfor treating neurodegenerative diseases.The chemical enhancement of CMA can protect cells from oxidative stress and proteotoxicity. 

Signaling through retinoic acid receptor alpha (RAR ) inhibits CMA activity.Synthetic derivatives of all-trans-retinoic acid can specifically neutralize this inhibitory effect [32]. Recently, it was also reported that human has molecular functions to antagonizeendogenous CMA inhibitors and promote interaction between the CMA chaperone Hsp90and the CMA receptor LAMP2A. 

Humanin and its analogs can enhance the CMA pathwayby increasing substrate binding and translocation into lysosomes and exerting cytoprotectiveeffects against hypoxia-induced cell death [130]. Another study found that metformin, adrug commonly prescribed for type 2 diabetes, can activate the CMA pathway and preventthe accumulation of amyloid- plaque in an animal model of AD [85]. 

Different protein degradation systems are wired to maintain cellular proteostasisunder various physiological and pathological conditions. Protein degradation via CMAis achieved through the lysosome-based autophagy system and therefore interacts withmacroautophagy and UPS [4,40,131]. Thus, a therapeutic approach that activates the CMA,macroautophagy, and UPS pathways should provide complementary or synergistic effectsin restoring protein homeostasis [40]. 

However, the molecular mechanism involved inthe interplay between these different protein degradation pathways has not been fullyelucidated. Exploring the mechanisms underlying the cross-talk between CMA, macroautophagy, and UPS may facilitate the development of an effective therapeutic strategy torestore proteostasis in various neurodegenerative diseases.

8. Therapeutic Potential of CMA for Acute Neurological Insults

Following acute neurological insults to the CNS, including cerebral infarction, TBI, andSCI, secondary injury can be induced by various molecular mechanisms, such as oxidativestress and neuroinflammation in the brain and spinal cord [132,133]. Such secondary injuryis involved in multiple pathologies associated with neural cell death and neurodegeneration,aggravating the initial tissue damage of the CNS [112,113,132]. 

The secondary damage canbe a potential therapeutic target for the effective treatment of acute neurological insults to theCNS. Many previous studies have shown that activation of the autophagic process can exerta neuroprotective effect against secondary damage after acute CNS injury [99,134]. Notably,several studies have suggested that the upregulation of CMA activity may help reducesecondary neural tissue damage following acute neurological insults to the CNS [6]. 

Asmentioned above, mycophenolic acid administered to activate the CMA pathway rescuedhypoxia-mediated cell death after brain ischemia in an in vitro model [17]. In addition,Tat-CDK5-CTM increases the CMA degradation of CDK5, reducing the infarction areaand neuronal loss and improving the neurological functions in a mouse model of cerebral infarction [106]. 

Furthermore, HDAC6 can regulate Hsp90 acetylation to enhance CMAactivity and exert a neuroprotective effect after SCI in mice [116]. The upregulation ofDnajb1 expression induced by Sirt1 activated CMA and consequently reduced neuronalloss in a mouse model of TBI [100]. 

Therefore, enhancing the CMA pathway to remove toxicproteins may be a novel therapeutic approach to reduce secondary neural tissue damageafter acute neurological insults.Acute neurological insults in the CNS damage different types of neural cells, such asneurons, oligodendrocytes, astrocytes, and microglia. Such damage to these neural cellscauses complex pathophysiological processes, including extensive neuronal cell loss, axonalinjury, demyelination, and destruction of the blood-brain/spinal cord barrier [132,133]. 

Importantly, the activity of CMA is increased not only in neurons but also in microglia atthe lesion site after TBI and SCI [16,18]. Microglia play various important roles in neuroprotection and neuroinflammation following acute CNS injury [135–137]. 

As described above,annexin A1 peptide enhances the CMA activity to degrade IKK and consequently reducesthe TNF- expression in microglia, suggesting an anti-inflammatory mechanism associatedwith CMA [101]. In addition, the activity of the CMA pathway is also upregulated inastrocytes and oligodendrocytes after SCI in mice [18]. 

CMA activation has been shownto reduce -synuclein accumulation in astrocytes and oligodendrocytes in vitro [138,139].The reduction in -synuclein aggregation in the injured spinal cord has been reported toprovide neuroprotective effects, attenuating axonal damage, neuronal loss, and neuroinflammation following SCI [140]. 

Previous studies have also suggested that autophagicactivity contributes to the survival of oligodendrocytes and the prevention of myelin loss afterSCI [141]. Taken together, modulation of CMA activity in various types of glial cells mayaffect multiple pathophysiological processes following acute neurological insults in theCNS. 

It is important to determine the molecular mechanisms underlying the interactionbetween CMA and various pathologies in the damaged CNS.Many studies have suggested that depositions of aberrant proteins, such as amyloid- and Tau protein, are observed in the brains of patients after TBI [142]. The pathologicalaccumulation of aberrant proteins after TBI can be a major risk factor for several progressiveneurodegenerative diseases, such as AD and PD [142,143]. 

The aggregation of amyloid- is accelerated in injured brains, and amyloid- plaques can be a pathological cause ofneurodegenerative diseases in chronic-stage TBI [144,145]. TBI can also reportedly inducethe aggregation of Tau proteins, which is a common feature of several neurodegenerativedisorders [146]. Importantly, enhancement of the CMA pathway can decrease the accumulation of amyloid- and Tau proteins in the brain [4,6,85,117,147]. Thus, the upregulationof CMA may aid in removing toxic aberrant proteins causing late-onset neurodegenerationafter TBI.

9. Concluding Remarks and Future Perspectives

In the past decade, the regulatory mechanisms involved in the CMA degradationpathway have become clearer, expanding our understanding of the importance of CMA incellular functions [4,6,8]. 

There is increasing evidence that CMA dysfunction is associatedwith different pathologies in neurodegenerative diseases in the CNS [1,4,6,14,15]. Importantpathogenic proteins have been identified as the substrates of CMA, such as -synucleinin PD [60], Tau protein in AD [61], huntingtin (Htt) in HD [62,63], and TDP-43 in ALSand FTLD [64,65]. 

However, most previous studies related to CMA in the CNS havefocused on neurodegenerative diseases rather than acute neurological insults, such asTBI and SCI [6,14]. 

The CMA function in acute neurological insults in the CNS is still animmature research field and limited evidence has been published thus far. As mentionedabove, CMA activity is likely to be upregulated in damaged neural tissues after acute CNSinjury [16,18,100,116]. The actual function of CMA activation following acute injury of thebrain and spinal cord remains unknown. Therefore, further studies will be necessary toassess the possible association of CMA with acute neurological insults in the CNS.

Various compounds have been reported to increase LAMP2A levels and activatethe CMA pathway [118–124], as described above. However, these compounds cannotselectively regulate the CMA pathway. 

Therefore, it is important to develop selectiveCMA modulators that can be used for clinical treatment of human diseases [4]. Thedevelopment of pharmacological selective CMA modulators will be a crucial step towardsthe implementation of therapeutic strategies aimed at improving cellular homeostasisthrough the regulation of CMA in the CNS. 

Several currently available FDA-approved drugs and natural products have beenfound to promote CMA activity [85,121,148,149]. These drugs and products that enhanceCMA might be able to be translated into novel clinical applications. Clinical trials involvingautophagy as a therapeutic target for neurodegenerative diseases have focused on macroautophagy, not CMA [150,151]. 

Author Contributions: Conceptualization, H.K. and K.H.; writing-original draft preparation, H.K.and K.H.; writing-review and editing, H.K. and K.H.; visualization, H.K., K.H., and T.M.; supervision,T.A. and H.O. All authors have read and agreed to the published version of the manuscript.

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