Chaperone-Mediated Autophagy in Neurodegenerative Diseases And Acute Neurological Insults in The Central Nervous System Part 2

4. Experimental Research Tools to Assay CMA Activity

In this section, we describe the experimental research tools available for assaying CMA activity in vitro and in vivo. The methods commonly used to obtain information correlated with CMA activity as well as CMA functional assays.

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4.1. Useful Analyses for Monitoring CMA Activity

Evaluating changes in the amounts of the main molecular components of CMA can be used as an indirect way of assessing CMA activity [46]. In addition, the number and distribution of CMA-active lysosomes can be analyzed to infer changes in CMA activity [46]. 

However, these analyses only provide the data correlated with CMA status. Thus, these analyses should be complemented with functional assays [47]. Immunoblotting and imaging for evaluating changes in the main CMA components are the most commonly used methods for assessing CMA activity. 

Since the lysosomal levels of LAMP2A are limited for CMA [48], changes in the abundance of the LAMP2A protein in lysosomes usually correlate with the activity of CMA. Therefore, in imaging evaluations, the presence of LAMP2A at the lysosomal membrane should be analyzed [47]. 

Immunoblotting for LAMP2A using lysosome-enriched fractions or at least a membranous cell fraction is more informative than that using total whole-cell lysates [46]. Levels of lysosomal-hsc70 are also related to CMA activity [49]. 

However, hsc70 is one of the most abundant cellular chaperones, and the fraction located in lysosomes is a small amount. Therefore, immunoblotting for hsc70 in total cellular lysates is not informative for CMA [46]. Colocalization of hsc70 with lysosomal markers (e.g., LAMP1) can be used to detect CMA-active lysosomes [49]. 

The number of these lysosomes colocalized with hsc70 in proportion to the whole lysosomal pool increases when CMA is activated [12]. In addition, an electron microscopic analysis using immunogold staining for hsc70 can also provide information about the pool of CMA-active lysosomes [49]. 

In analyses using isolated lysosomes from cells or tissues, increased levels of well-known CMA substrates (e.g., GAPDH) can indicate a decreased activity of CMA [46]. 

In general, CMA substrates are rapidly degraded after translocation [6]. Thus, a comparison of the lysosomal levels of CMA substrates in cells or animals between models treated or untreated with inhibitors of lysosomal proteases (i.e., leupeptin) allows for the measurement of the flux in CMA values [24].

4.2. Functional Assays

Several functional assays enable the tracking of CMA activity over time in cells, tissues, and isolated organelles.

4.2.1. Intracellular Protein Degradation Assessment

Approximately 30% of total cytosolic proteins can be degraded by CMA [9]. However, the actual fraction of the cytosolic protein degraded by CMA varies depending on the cell type and cellular conditions [6]. 

Therefore, measurement of the pool of cellular proteins that undergo degradation through CMA is a common method for determining the overall activity of the CMA pathway [46]. 

Pulse and chase experiments using a radiolabeled amino acid and inhibitors of either lysosomal proteases or other autophagic pathways can be used to discriminate proteins undergoing CMA degradation from those managed by other pathways [50].

4.2.2. Photoconvertible CMA Reporters

In addition, a method of monitoring the lysosomal association of artificial fluorescent CMA reporters would also be useful for tracking substrate delivery and degradation through CMA [51]. 

Using photoconvertible fluorescent reporters [51], it is possible to track the association of the photoconverted protein with lysosomes in a different fluorescence channel. An increase in the number of fluorescent puncta per cell would be a good indicator of CMA activation [46].

4.2.3. In Vitro Analyses of CMA Using Isolated Lysosomes

The cross-talk between different autophagic pathways makes it difficult to accurately assess CMA activity in intact cells [41]. Therefore, we must separately analyze all functional steps involved in the dynamic degradation process of CMA pathways [46,49]. 

The most reliable approach for analyzing CMA activity is obtained by in vitro reconstitution of CMA with isolated lysosomes [52]. Isolation of the specific fraction of lysosomes active in CMA allows for an analysis of the content of endogenous CMA substrates in the CMA compartments [47]. 

Isolated lysosomes also permit the reconstitution of CMA in vitro to follow the steps involved in the CMA process-substrate binding, lysosomal uptake, and lysosomal degradation [50]. 

Treatment with lysosomal protease inhibitors followed by incubation with the CMA substrate will allow for the measurement of the substrate-bound and translocated into lysosomes (binding and uptake) [39]. 

By discounting the amount of substrate bound to lysosomes in which proteolysis has not been prevented, it would then be possible to calculate the uptake [39,53]. 

The isolated lysosomal fractions also allow for the direct comparison of changes in the content, post-translational modification, and organization of CMA components at the lysosomal membrane. Reductions in lysosomal LAMP2A or lys-hsc70 levels in isolated lysosomes are indicative of decreased CMA activity [54,55], whereas increases in LAMP2A levels suggest upregulation of CMA activity [56]. 

In addition, the ratio of lysosomal LAMP2A assembled into a multimeric complex at a given time can be determined using blue native electrophoresis of isolated lysosomes and immunoblot for LAMP-2A [57].

5. Neurodegenerative Diseases and CMA

Neurons are post-mitotic cells and require efficient protein degradation machinery to maintain cellular homeostasis under stress conditions [58,59]. Impairment of the protein degradation process in the CNS causes aggregation of aberrant or damaged proteins, which is a distinct feature of many neurodegenerative diseases. 

Substantial evidence has been gathered that dysfunction of CMA is associated with different pathologies in various neurodegenerative diseases affecting the CNS [1,14]. 

In these diseases, various pathogenic proteins have been identified as the substrates of CMA, such as α-synuclein in PD [60], Tau protein in AD [61], huntingtin (Htt) in HD [62,63], and TDP-43 in ALS and FTLD [64,65].

5.1. Parkinson's Disease

PD is one of the most common neurodegenerative disorders. The main pathological features of PD are the gradual loss of dopaminergic neurons within the substantia nigra and aggregation of the protein α-synuclein in Lewy bodies. 

Numerous studies have shown that impairment of CMA is related to the main pathogenesis of PD [4,66]. In patients with PD, the level of LAMP2A protein is decreased in the brain, indicating that CMA activity is attenuated [67,68]. 

Many previous studies have suggested that inhibition of the CMA degradation pathway causes the accumulation of α-synuclein, which is associated with the gradual loss of dopaminergic neurons [8]. 

Importantly, the mutant forms A53T and A30P of α-synuclein identified in familial PD cannot be degraded by CMA. Furthermore, these mutant forms tightly bind to LAMP2A at the lysosomal membrane and consequently inhibit the normal degradation of other CMA substrates in vitro [60,69]. G2019S mutation in leucine-rich repeat kinase 2 protein (LRRK2) can be a pathological cause of familial PD [70]. 

The G2019S mutant inhibits the dynamic assembly of the CMA translocation complex at the lysosomal membrane, causing the dysfunction of CMA in a mouse model of PD and the brains of mutant LRRK2 PD patients [25]. In addition, the pathogenic mutant forms of LRRK2 bind to cytosolic Hsc70 and interact abnormally with CMA components, blocking the degradation of other CMA substrates and neuronal protein homeostasis in vitro [25,71]. 

Ubiquitin C-terminal hydrolase L1 (UCH-L1) physically interacts with LAMP-2A, Hsc70, and Hsp90 and is involved in the regulatory mechanism of the CMA pathway [72]. In a previous study, the I93M mutant form of UCH-L1 was identified in a single PD family [73]. 

It has also been reported that the I93M mutation in UCH-L1 abnormally enhanced interaction with the cytosolic region of LAMP2A, inhibiting the CMA pathway in vitro [74]. Furthermore, the expression of the I93M mutant form of UCH-L1 in mammalian cells induced the CMA inhibition-associated increase in the amount of αsynuclein [74]. 

These findings suggest that aberrant interaction of the I93M mutant form of UCH-L1 with CMA machinery might underly the pathogenesis of PD associated with the aggregation of α-synuclein. Parkinson's disease protein 7 (PARK7), also known as DJ-1, is a multifunctional protein involved in a variety of cellular activities, including oxidation resistance [75]. 

PARK7/DJ-1 has an important role in maintaining mitochondrial homeostasis [75]. It has been reported that a mutation in the DJ-1 gene mediates autosomal recessive and early forms of PD [76]. DJ-1 deficiency accelerated the degradation of LAMP2A in lysosomes, leading to the aggregation of α-synuclein [77]. 

In contrast, DJ-1 was able to inhibit the accumulation of α-synuclein by regulating CMA [78]. Overall, various molecular mechanisms causing dysfunction of CMA are considered to underlie the pathogenesis of PD. 

However, the pathological mechanisms associated with CMA remain largely unclear. Further research will be needed to clarify the relationship between the CMA process and the actual pathologies of PD.

5.2. Alzheimer's Disease

AD is the most common neurodegenerative disease in the elderly. The main pathogenesis of AD is amyloid-β plaque formation and Tau aggregation caused by the impairment of protein homeostasis. Several proteins related to AD have been identified as CMA substrates. The CMA degradation of these protein substrates was shown to be impaired in patients with AD [45,61,79]. 

The progressive accumulation of amyloid-β oligomers is a central toxic event in AD [80,81]. A recent study showed that tagging amyloid-β oligomers with multiple KFERQ motifs promoted their entry into endosomes and lysosomes, protecting human primary cultured cortical neurons from neurotoxicity [82]. Amyloid precursor protein (APP) is an important pathogenic molecule in AD because it can be processed to produce amyloid-β [83]. 

APP contains a KFERQ-like motif at its C terminus. This motif is important for the normal processing and degradation of APP to prevent the accumulation of APP-C-terminal fragments [84]. A recent study revealed that APP is a CMA substrate that binds to Hsc70 [85]. 

The inhibition of CMA degradation of APP enhances its cytotoxicity. Furthermore, activation of CMA by Hsc70 overexpression or Metformin reduced the accumulated brain amyloid-β plaque levels and reversed the molecular and behavioral AD phenotypes in a mouse model of AD [85]. Tau is a cytosolic protein that normally stabilizes microtubules in neuronal cells. 

Tau protein has CMA-targeting motifs and can be degraded by the CMA pathway [79]. Aggregation of mutant Tau proteins resulting in Tau hyperphosphorylation and the formation of neurofibrillary tangles is a hallmark of AD and related tauopathies [86,87]. 

In addition, the mutant Tau proteins can interact abnormally with LAMP2A and inhibit translocation into the lysosome lumen, impairing CMA activity [79]. The regulator of calcineurin 1 (RCAN1) is a substrate of CMA [45] and is elevated in patients with AD [88]. 

RCAN1 is an inhibitor of calcineurin-dependent dephosphorylation of Tau proteins. Importantly, CMA activity can be inhibited by increasing the level of RCAN1, thereby impairing the degradation of other substrates of CMA [45].

5.3. Huntington's Disease

HD is a late-onset neurodegenerative disorder characterized by uncontrolled movement, dementia, and emotional disturbance. HD is a dominantly inherited disease caused by the accumulation and aggregation of mutant Htt protein in striatal and cortical neurons. 

Htt contains an abnormally expanded N-terminal polyglutamine (polyQ) tract [62,63,89]. Dysfunction of Htt degradation is suggested as the main pathogenesis of HD. 

Previous studies have shown that CMA is involved in the degradation of mutant Htt in cellular and mouse models of HD [62]. Htt harbors a putative KFERQ motif and interacts with the key components of CMA, Hsc70, and LAMP2A. In addition, mutant Htt with an expansion of the polyQ tract displays an impaired uptake by CMA in vitro [89]. 

Not only CMA but macroautophagy is involved in the degradation of Htt [90]. Htt can bind to both LAMP2A and the macroautophagy-related protein Atg7 in the degradation process [89,91,92]. 

CMA activity is reportedly upregulated in cellular and animal models of HD in the initial stage of the disease. However, the activity of CMA decreases during the late stage of the disease [91]. These findings suggest that the early increase in CMA activity may be a compensatory regulation in response to the inefficiency of macroautophagy. The decline in the level of lysosomal LAMP2A indicates that there is a loss of CMA function in the late phase of HD [91].

5.4. Amyotrophic Lateral Sclerosis and Frontotemporal Lobar Degeneration

ALS and FTLD are neurodegenerative diseases with many similar clinical and pathological features [93]. Transactivation response DNA-binding protein 43 kDa (TDP-43) is a ribonuclear protein regulating many aspects of RNA metabolism. 

The accumulation of TDP-43 C-terminal fragments in neuronal cells is frequently detected in patients with ALS and FTLD [65]. Thus, TDP-43 accumulation is widely considered a hallmark of these diseases. 

TDP-43 protein contains a KFERQ-like motif binding to Hsc70 and can be degraded by the CMA process [94,95]. Hsc70 expression was reportedly reduced in lymphomonocytes of sporadic ALS patients and contributed to TDP-43 accumulation [64]. 

A mutation in the KFERQ-like motif in TDP-43 can disrupt its degradation via CMA, inducing the accumulation of TDP-43 and the inhibition of CMA in cultured cells [94]. CMA can help control the turnover of the physiological and pathological forms of TDP-43 [94].

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