A role of Heat Shock Protein 70 in Photoreceptor Cell Death: Potential as a Novel Therapeutic Target in Retinal Degeneration

Summary

Retinal degenerative diseases (RDs) such as retinitis pigmentosa (RP) are a genetically heterogeneous group of disorders characterized by night blindness and peripheral vision loss, which caused by the dysfunction and death of photoreceptor cells. Although many causative gene mutations have been reported, the final common end stage is photoreceptor cell death. Unfortunately, no effective treatments or therapeutic agents have been discovered. Heat shock protein 70 (HSP70) is highly conserved and has antiapoptotic activities. A few reports have shown that HSP70 plays a role in RDs. Thus, we focused on the role of HSP70 in photoreceptor cell death. Using the N‐methyl‐N‐nitrosourea (MNU)‐induced photoreceptor cell death model in mice, we could examine two stages of the novel cell death mechanism; the early stage, including HSP70 cleavage through protein carbonylation by production of reactive oxygen species, lipid peroxidation and Ca²⁺ influx/calpain activation, and the late stage of cathepsin and/or caspase activation. The upregulation of intact HSP70 expression by its inducer is likely to protect photoreceptor cells. In this review, we focus on the role of HSP70 and the novel cell death signaling process in RDs. We also describe candidate therapeutic agents for RDs.

Introduction

Retinitis pigmentosa (RP) is one of the major retinal degenerative diseases (RDs), which are caused by photoreceptor cell death 1. At least 50 million people have these diseases, and no effective drugs have been discovered. Animal models of RP have led to a better understanding of the disease pathology and to the development of therapeutic strategies aimed at curing or slowing down the genetic disorder 2. It is not easy to choose an appropriate genetic model for RP because there are many causative genes 3; more than 30 genes and more than 100 rhodopsin mutations are related to RP. Although animal models of RP have a variety of genetic backgrounds (Table 1) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, the final common end stage of RP is photoreceptor cell death.

Table 1.

Genetic models for RP

Animal models		Genotypes	Genes	Site of origin	References
Mice	Natural	Peripherin‐rds	Peripherin‐rds	Null mutation in the rds/peripherin gene	4
		Rd	Peripherin‐rds	rd/rd (retinal degeneration) mice	5
		Rd‐1	PDE6B	Nonsense mutation in exon 7 of the Pde6b gene in all mouse strains with the rd1 mutation	6
		Rd‐4	–	Inversion encompasses nearly all of Chromosome 4	7
		Rd‐8	CRB1	Single base deletion in the Crb1 gene	8
		Rd‐10	PDE6B	Mutation in PDE6b	9
		Rd‐12	RPE65	Homozygous for the rd12 mutation	10
		Rd‐16	CEP290	In‐frame deletion in a centrosomal protein CEP290	11
	Transgenic	307 1‐bp del	Peripherin‐rds	Single base deletion at codon 307 of the rds‐peripherin gene in mice	12
		C214S	Peripherin‐rds	Peripherin‐rds with the C214S (Cys214–>Ser) missense mutation	13
		Crx knockout	Cone‐rod homeobox	Cone‐rod homeobox gene knockout	14
		Knockout RPE65	Rhodopsin	Mice that lack the visual pigment rhodopsin (Rpe65‐/‐)	15
		l‐255/256	Opsin	Mutant opsin gene with a 3‐bp deletion of isoleucine at codon 255/256	16
		L185P/Rom‐1 null	Peripherin‐rds	Doubly heterozygous for a mutation in RDS causing a leucine 185 to proline substitution in rds (L185P) and a null mutation in ROM1	17
		MERTK KO	MERTK	Homozygous for a targeted disruption of the Mer receptor tyrosine kinase gene (mer(kd))	18
		NMF282	PDE6A	Ethyl nitrosourea (ENU) mutagenesis	19
		NMF363	PDE6A	Ethyl nitrosourea (ENU) mutagenesis	19
		P216L	Peripherin‐rds	Proline 216 to leucine (P216L) amino acid substitution in rds/peripherin	20
		P23H	Rhodopsin	Missense mutation (P23H) in the rhodopsin gene	21
		P347S	Rhodopsin	Rhodopsin, proline‐347 to serine (P347S) mutation	22
		Q344ter	Rhodopsin	Heterozygotes with the glutamine‐344‐to‐ter (Q344ter) mutations in the rhodopsin gene (stop codon mutation)	23
		Rd12j (NMF137)	PDE6B	Missense point mutation in exon 16 of the beta‐subunit of rod phosphodiesterase gene, (PDE6B)	9
		Rpe65−/−	RPE65	Rpe65‐deficient (KO)	24
		Sema4A‐deficient	Sema4A	Sema4A‐deficient	25
		Sema4A F350C	Sema4A	Knock‐in mouse lines with corresponding mutations (F350C) in the Sema4A gene	25
Rat	Natural	RCS	MERTK	Small deletion of RCS DNA that disrupts the gene encoding the receptor tyrosine kinase Mertk	26
	Transgenic	P23H	Rhodopsin	Transgenic rat that express P23H rhodopsin	27
		S334ter	Rhodopsin	Rhodopsin mutation S334ter	28
Chickens	Natural	Rd	GC1	Null mutation in the photoreceptor guanylate cyclase (GC1) gene	29
		Rdd	PDE6A	Mutation in PDE6A	30

N‐Methyl‐N‐nitrosourea (MNU), an alkylating agent, causes photoreceptor cell loss and significantly decreases the outer nuclear layer thickness within 1 week after intraperitoneal injection 31, 32, 33. MNU selectively damages photoreceptor cells; no other retinal cells are TUNEL positive. Thus, we used the MNU model to study the mechanism of photoreceptor cell death.

Heat shock protein 70 (HSP70) plays an important role in protecting cells against various stresses. However, a few reports have shown the effect of HSP70 on photoreceptor cell death in RDs. In the present review, we describe the role of HSP70 in photoreceptor cell death and discuss the possibility of HSP70 inducers as a new therapeutic tool for RDs.

The Mechanisms of MNU‐Induced Photoreceptor Cell Death

Some reports have suggested that MNU induced the generation of free radicals and cell death specifically in retinal photoreceptor cells. Accumulation of 8‐hydroxy‐deoxyguanosine, an indicator of oxidatively damaged DNA, and 4‐hydroxy‐2‐nonenal (4HNE), a reactive aldehyde species generated endogenously from decomposition of hydroperoxide of ω‐6 polyunsaturated fatty acids 34, was detected in MNU‐treated mouse retina 33, 35. MNU also causes a decrease in reduced glutathione, which effectively scavenges free radicals and other reactive oxygen species (ROS) 36, leading to an imbalance between the production of ROS and antioxidants.

Intraperitoneal injection of MNU induces the accumulation of intracellular Ca²⁺ in the retina and increases calpain activation, as measured by α‐spectrin proteolysis products, which leads to photoreceptor cell death 37, 38.

MNU‐induced photoreceptor cell loss is caused by a decrease in antiapoptotic Bcl‐2 protein, an increase in proapoptotic Bax protein, and the activation of caspase cascades 39, 40. Caspase‐3, caspase‐6, and caspase‐8 activities were increased within 3 days after MNU injection.

Although such molecular mechanisms of the MNU‐induced photoreceptor cell loss have been described, the total process of the cell death signaling remains obscure. Elucidation of the key molecule that connects these molecular mechanisms is necessary to clarify the photoreceptor cell death signaling process.

Early and Late Stages of MNU‐Induced Photoreceptor Cell Death Processes

The HSP70 family is a family of conserved and ubiquitously expressed heat shock proteins. HSP70 is a central component of the cellular network of molecular chaperones and folding catalysts and protects cells from various stresses. Although HSP70 immunoreactivity is localized in the outer nuclear layer and the inner segments of the retina 41, a few studies have reported the role of HSP70 in RDs. Thus, we investigated the role of HSP70 on MNU‐induced photoreceptor cell death 33. Under pathological conditions of neuronal tissues, such as glaucoma and ischemic/reperfusion of the hippocampus, HSP70 is a common substrate of calpain 42. Carbonylated HSP70 by 4HNE is much more vulnerable to calpain cleavage 43. We found that the levels of 4HNE were clearly increased in MNU‐injected mouse retina. 4HNE is highly reactive and may be considered as a secondary toxic messenger that disseminates and augments initial free radical events 44, 45. Upon the reaction with protein, 4HNE specifically reacts with nucleophilic amino acids, such as cysteine, histidine, and lysine to form their Michael addition adducts possessing carbonyl functionality 46, 47. Thus, HSP70 may be carbonylated by the accumulated 4HNE in MNU‐treated mouse retina. In addition, we confirmed that HSP70 was rapidly and calpain‐dependently cleaved after MNU treatment. Our results indicate that HSP70 cleavage might be involved in both oxidative stress and Ca²⁺/calpain‐mediated photoreceptor cell loss. Calpain‐mediated cleavage of HSP70 leads to lysosomal rupture and cell death through cathepsin because HSP70 stabilizes lysosomal membranes 48; this process is known as the calpain–cathepsin hypothesis 49, 50. On the other hand, HSP70 protects against neuronal apoptosis through the inhibition of caspase‐dependent apoptosis 51, 52. Thus, caspase‐dependent apoptosis occurs in downstream of HSP70 cleavage. Together, our findings suggest that cleavage of HSP70 is a key event that connects the mechanisms of MNU‐induced photoreceptor cell death (Figure 1). Focusing on HSP70, the MNU‐induced cell death signaling process can be divided into early and late stages. We defined the early stage as HSP70 cleavage through protein carbonylation by oxidative stress, 4HNE production, and Ca²⁺/calpain activation. The late stage includes the events after HSP70 cleavage, including cathepsin and/or caspase activation.

Figure 1.

The mechanism of MNU‐induced photoreceptor cell death and the candidate therapeutic agents for RDs.

HSP70 Induction Prevented Photoreceptor Cell Death by MNU

To determine whether HSP70 could protect photoreceptor cell death by MNU, we used valproic acid (VPA), a well‐known HSP70 inducer 53. VPA significantly inhibited MNU‐induced retinal thinning and TUNEL‐positive photoreceptor cell number through HSP70 induction. Coadministration of VPA and HSP inhibitor abolished the protective effect of HSP70; thus, HSP70 plays a crucial role in the protection of photoreceptor cells. Calpain inhibitor also protects photoreceptor cells because of the suppression of HSP70 cleavage. VPA failed to protect HSP70 from MNU‐induced cleavage, but increased the expression levels of intact HSP70. Both VPA and calpain inhibitor completely blocked caspase‐3 activation by MNU. In addition, we previously reported that geranylgeranylacetone (GGA), another HSP70 inducer, also attenuated the photoreceptor cell death by MNU through HSP70 induction 33, 54. Thus, inhibition of HSP70 cleavage or induction of intact HSP70 may be possible therapeutic approaches for preventing photoreceptor cell death.

Cytoprotective Effects of HSP70

The photoreceptor cell layer is the primary site of HSP70 synthesis in the retina, and hyperthermia‐induced HSP70 in the photoreceptor layer prevents retinal photic injury 55, 56. In the retinal detachment‐induced retinal degeneration model, abolishment of HSP70 induction using HSP70^−/− mice directly exacerbated photoreceptor apoptosis 57. Furthermore, HSP990, a HSP70 inducer, enhanced visual function and delayed photoreceptor degeneration in a rhodopsin mutation rat model 58. These results are in accordance with previous in vitro and in vivo studies that showed that abolishment of the HSP70 cytoprotective effect augments the initiation of the apoptotic cascade 59, 60, 61. Even in the CNS model, previous studies have reported extensive neuronal damage in HSP70^−/− mice after ischemic brain injury, in which the neuronal expression of HSP70 can be regarded as a molecularly defined penumbra of protein denaturation 62. Thus, HSP70 overexpression directly increased the neuronal viability in various CNS degeneration models 63, 64. These reports showed that HSP70 directly prevented photoreceptor cell death in both genetic RP models and acquired models.

Candidate Therapeutic Agents for RDs

Based on the total image of photoreceptor cell death mechanism that we proposed, protein carbonylation by oxidative stress, Ca²⁺‐dependent protease activation, apoptosis‐related molecules, and HSP70 cleavage are involved in MNU‐induced photoreceptor cell death. The final common end stage of various pathogenic mechanisms in RDs is photoreceptor cell death. Therefore, protection of photoreceptor cells may be a useful therapeutic strategy for RDs. Some of the candidate therapeutic agents for RDs are listed in Table 2 [33, 35, 37, 39, 54, 58, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121]; these agents include therapeutic agents against the early stage of photoreceptor cell death (antioxidants, ROS scavengers, Ca²⁺ antagonists and calpain inhibitors), therapeutic agents against the late stage of photoreceptor cell death (cathepsin inhibitors and caspase inhibitors), and HSP70 inducers.

Table 2.

Candidate therapeutic agents for RDs

Roles	Compounds	References	Roles	Compounds	References
ROS scavenger	5‐S‐GAD	65	HSP70 inducer	17‐AAG	92
/Antioxidant	Alpha lipoic acid	66		17‐DMAG	92
	Astaxanthin	67		2‐Cyclopenten‐1‐one	93
	Carnosic acid	68		Alkannin	94
	Curcumin	69		Arimoclomol	95
	DHA	70		Bicyclol	96
	Edaravone	35		Bimoclomol	97
	Green tea extract	71		Carbenoxolone	98
	Lutein	72		CdCl2	99
	Melatonin	73		Celastrol	100
	N‐acetylcysteine	74		Curcumin	101
	Unoprostone	75		Eupalinolide A/B	102
				FLZ	103
Ca chelator/	2‐APB	76		Geldanamycin	104
/Ca blocker	BAPTA‐AM	77		GGA	54
	Diltiazem	78		Glucuronic acid	105
	Flunarizine	79		HSP990	58
	Nicardipine	80		KU‐32	106
	Nilvadipine	81		Linolenic acid	107
	Nimodipine	36		MG132	99
				Paeoniflorin	108
Calpain inhibitor	ALLN	82		Polaprezinc	109
	Calpastatin	83		Prostaglandin A1	110
	Calpeptin	77		Radicicol	111
	CYLA	84		Rebamipide	112
	MDL28170	85		Resveratrol	113
	MG132	86		Safrole oxide	114
	PD150606	82		Sodium butyrate	115
	SJA6017	87		Sodium fluoride	116
	SNJ‐1945	37		Sodium salicylate	117
				TRC051384	118
Cathepsin inhibitor	CA‐074Me	88		Tributyltin	119
	E‐64	89		VPA	33
	Z‐FA‐FMK	90		YC‐1	120
	Z‐FY(t‐Bu)‐DMK	91		Zinc	121
Caspases inhibitor	Ac‐DEVD‐CHO	39

As therapeutic agents against the early stage of photoreceptor cell death, antioxidants and ROS scavengers could prevent HSP70 carbonylation through reduction of 4HNE production. In MNU‐injected mouse retina, edaravone, a ROS scavenger, can reduce 4HNE generation and the number of TUNEL‐positive cells 35. Polyphenols, such as curcumin and green tea extract, also reduced the number of MNU‐induced TUNEL‐positive photoreceptor cells 69, 71. The Ca²⁺ antagonist and calpain inhibitors could also protect photoreceptor cells via the inhibition of HSP70 cleavage. Nimodipine, a Ca²⁺ blocker, inhibits MNU‐induced photoreceptor cell apoptosis and protects retinal function 36. A calpain inhibitor, SNJ‐1945, restored photoreceptor cell autophagy and photoreceptor cell death in MNU‐treated mice 37, 38.

Furthermore, as therapeutic agents against the late stage of photoreceptor cell death, both inhibitors of cathepsin and caspases could suppress photoreceptor cell death. Caspase inhibitor was shown to suppress retinal apoptosis in MNU‐treated rats 39.

In addition to these existing therapies, we further propose that HSP70 inducers could be novel therapeutic agents to prevent photoreceptor cell death in RDs. Many different chemicals have been reported as HSP70 inducers, including arimoclomol 95, celastrol 100, eupalinolide A/B 102, paeoniflorin 108, and radicicol 111 (Table 2). Drug repositioning is the process of developing new indications for existing drugs or biologics. Some antiulcer agents, such as carbenoxolone 98, polaprezinc 109, and rebamipide 112, induce HSP70 expression in various human tissues. Thus, the drug repositioning approach by HSP70 inducers could be an effective way to develop new therapeutic agents for RDs.

Similar Mechanisms of Photoreceptor Cell Death between MNU and RP Models: Early Stage

Although the MNU‐induced photoreceptor cell death model is different from the genetic RP model, the two models appear to share similar mechanisms of photoreceptor cell death. The eye has 3‐ to 4‐fold higher oxygen consumption relative to brain tissue and, consequently, has a higher exposure to ROS such as hydrogen peroxide, hydroxyl radicals, and superoxide anions. Oxidative stress is involved in the pathogenesis of a number of diseases including neurodegenerative disorders such as RP 35, 122. Orally administered N‐acetylcysteine reduced photoreceptor cell death and preserved cone function by reducing oxidative damage in two models of RP, rd1, and rd10 mice, which have a mutation in the rod photoreceptor‐specific cGMP phosphodiesterase (PDE) subunit 74. In addition, coexpression of superoxide dismutase 2 and catalase in the mitochondria of photoreceptors strongly promotes cell survival and the maintenance of photoreceptor function in rd10 mice 123. In some RP models, accumulation of 4HNE was detected in photoreceptor cells 124, 125. Therefore, oxidative stress plays a pivotal role in genetic RP models of retinal photoreceptor degradation.

Under pathological conditions, like those in rd1 mice, intracellular Ca²⁺ levels significantly increase in photoreceptor cells, even before the detection of apoptotic cells 126. Increased photoreceptor cell death in the rd10 mouse retina is associated with Ca²⁺ overload and calpain activation, which both occur prior to signs of cell degeneration 127. Mitochondrial calpain may activate apoptosis‐inducing factor to induce photoreceptor apoptosis in Royal College of Surgeon (RCS) rats, a natural model of recessively inherited RDs that has a disrupted gene for the receptor tyrosine kinase 82, 128, and rhodopsin transgenic rats 129. μ‐Calpain contributed to the activation of Bax and apoptosis‐inducing factor nuclear translocation in rd1, P23H (missense mutation in the rhodopsin gene), and rhodopsin knockout retinas 130. The Ca²⁺ antagonist nilvadipine preserved retinal morphology and electroretinogram responses in RCS rats through the upregulation of fibroblast growth factor‐2 and antiapoptotic molecules in the retina 131. A small clinical trial revealed that nilvadipine retarded the progression of central visual field defects in RP 132. In addition to Ca²⁺ antagonists, calpain inhibitors can attenuate photoreceptor cell death. Mitochondrial μ‐calpain inhibitor prevents photoreceptor cell death in RCS rats 128. In rd1 mice, a highly specific calpain inhibitor, calpastatin, reduced photoreceptor cell death 133. Therefore, Ca²⁺‐dependent calpain activation may play an important role in RP and even in the MNU‐induced photoreceptor cell death model.

Similar Mechanisms of Photoreceptor Cell Death between MNU and RP Models: Late Stage

Cathepsin D also contributed to photoreceptor cell death in rd1, P23H, and rhodopsin knockout retina 130. Thus, cathepsin inhibitors may attenuate photoreceptor cell death.

In some RP models, altered expression of apoptosis‐related proteins was also involved in photoreceptor cell death 134. Inhibitors of caspase‐3, caspase‐7, and caspase‐9 also showed neuroprotection of photoreceptors at both the structural and functional levels in rhodopsin transgenic rat models of RP 135. Thus, caspase inhibitors are thought to be effective therapeutic tools for RP.

On the basis of similarities between genetic models of RP and MNU‐induced photoreceptor cell death, the therapeutic agents for MNU‐induced photoreceptor cell death might be effective in genetic models of RP.

Conclusion

In our recent studies, HSP70 carbonylation by 4HNE and its subsequent cleavage by calpain was one of the novel central mechanisms in photoreceptor cell death. In addition, VPA and GGA protected against photoreceptor cell death by MNU via the induction of HSP70 expression 33, 54. Further studies are needed to confirm these possibilities and to clarify the possible mechanism of pathogenesis and interaction between HSP70 cleavage and chronic photoreceptor cell death using a genetic model for RDs. Taken together, HSP70 inducers may be considered as candidate therapeutic agents for the prevention of RDs, such as RP.

Conflict of Interest

The authors declare no conflict of interest.