Potential Role of MANF, an ER Stress Responsive Neurotrophic Factor, in Protecting Against Alcohol Neurotoxicity

Abstract

Alcohol exposure during pregnancy is harmful to the fetus and causes a wide range of long-lasting physiological and neurocognitive impairments, collectively referred to as fetal alcohol spectrum disorders (FASD). The neurobehavioral deficits observed in FASD result from structural and functional damages in the brain, with neurodegeneration being the most destructive consequence. Currently, there are no therapies for FASD. It is exigent to delineate the underlying mechanisms of alcohol neurotoxicity and develop an effective strategy of treatment. ER stress, caused by the accumulation of unfolded/misfolded proteins in the ER, is the hallmark of many neurodegenerative diseases, including alcohol-induced neurodegeneration. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a newly discovered endoplasmic reticulum (ER) stress responsive neurotrophic factor that regulates diverse neuronal functions. This review summarizes the recent findings revealing the effects of MANF on the CNS and its protective role against neurodegeneration. Particularly, we focus the role of MANF on alcohol-induced ER stress and neurodegeneration and discuss the therapeutic potential of MANF in treating alcohol neurotoxicity such as FASD.

Introduction

Neurotrophic factors (NTFs) are small, secreted proteins or peptides that regulate the growth and survival of both developing and mature neurons. Three major classes of NTFs have been identified: (1) the neurotrophin family, consisting of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophins (NTs); (2) the glial cell line-derived neurotrophic factor family of ligands (GFLs), including glial cell line-derived neurotrophic factor (GDNF) and related growth factors; and (3) neuropoietic cytokines (neurokines), such as ciliary neurotrophic factor (CNTF), interleukin 6 (IL-6), and neuropoietin [1]. Upon secretion, each binds to cell membrane receptors to trigger various intracellular signaling pathways that promote neuron proliferation, differentiation, and migration, as well as support neuron survival and synaptic function [2, 3]. Recently, a new NTF, mesencephalic astrocyte-derived neurotrophic factor (MANF), has been discovered from a rat mesencephalic astrocyte cell to have neuroprotective effects on dopaminergic neurons [4]. Using bioinformatics and biochemical approaches, a MANF paralogue named cerebral dopamine neurotrophic factor (CDNF) was identified by sharing a high degree of sequence similarity with MANF [5]. CDNF and MANF thus form a novel class of NTFs. MANF is considered a non-canonical NTF as its protein structure is distinct from other NTFs [6], and it is mainly retained intracellularly within the endoplasmic reticulum (ER) under normal conditions, being secreted only in pathological conditions in which ER stress is induced [7-9]. MANF has been predicted to have two modes of actions. The first, like with conventional neurotrophic factors, when MANF is added or injected into extracellular space in both in vitro and in vivo models, it facilitates neuron survival in various pathological conditions, probably through the binding of cell membrane receptors and the activation of downstream pro-survival signaling pathways [10-12]. The second, as an ER resident protein, intracellular MANF protects neurons against Bax-dependent apoptosis, suggesting its distinct function inside cells [13].

MANF is widely expressed in the developing and mature central nervous system (CNS) [14-16]. Although MANF is regarded as a neurotrophic factor that plays an important role in the homeostasis of the mature CNS, it is also an important player in the regulation of CNS development. Embryonic MANF deficiency in Drosophila and zebrafish results in degeneration of dopaminergic neurons [17, 18]. In vitro and in vivo studies using neuronal cell cultures and MANF-deficient mice indicate a requirement of MANF for neurite outgrowth, neuronal differentiation, and cortical neuron migration in the developing brain [19, 20].

Alcohol is a commonly used substance. According to the 2019 National Survey on Drug Use and Health (NSDUH), about 70% of people age 18 or older reported alcohol consumption within the past year and more than 25% of adults reported that they had engaged in binge drinking (consume more than 4 drinks for women and 5 drinks for men in about 2 hours) within the last month [21]. Despite public education efforts and a suggested guideline to avoid alcohol consumption when pregnant, the global prevalence of alcohol use during pregnancy remains high [22-25]. The percentage of pregnant women consuming alcohol in the USA has increased steadily over time [25-28]. Excessive alcohol intake is neurotoxic and causes profound damage to the nervous system, affecting the structure, physiology, and function of both the mature and developing brain [29]. The developing brain is especially vulnerable to alcohol. Prenatal alcohol exposure during pregnancy can cause a wide range of long-lasting physiological and neurocognitive impairments, collectively referred to as fetal alcohol spectrum disorders (FASD). FASD are currently the leading cause of preventable intellectual disabilities in the USA. The prevalence of FASD is high and according to the National Institute on Alcohol Abuse and Alcoholism (NIAAA), 1–5 % of first-grade children in the USA have a FASD [30]. The most severe form of FASD is fetal alcohol syndrome (FAS), characterized by the malformation of the nervous system, cognitive impairments, abnormal cranial-facial development, and growth deficiency [31, 32]. Neuronal loss and neurodegeneration are the most devastating consequences of developmental exposure to alcohol [33, 34].

The mechanisms underlying alcohol-induced brain damage and neurodegeneration are poorly understood. Neuroinflammation [35, 36] and reactive oxygen species (ROS)-induced oxidative stress have been postulated to play an important role in alcohol neurotoxicity [37-39]. In addition, alcohol can alter the levels of neurotransmitters such as dopamine, glutamate, and serotonin as well as their receptors in the brain, contributing to the neuroapoptotic effects of alcohol upon the developing brain [40, 41]. Alcohol also disrupts the action of neurotrophic factors, which is considered a mechanism for its impact on the CNS [42]. Recent results suggest that ER stress is also involved in alcohol-induced neurodegeneration in both the developing and mature CNS [43-47].

ER stress is indicative of neurodegenerative diseases. It has been reported in various neurological disorders and injuries including Parkinson’s disease (PD), Alzheimer’s disease (AD), cerebral ischemia, spinal cord injury, sclerosis, and diabetic neuropathy [48-53]. The ER is an organelle important for the biological function and homeostasis of cells. It is responsible for the posttranslational modification of proteins and the synthesis of membrane lipids such as cholesterol. Many disturbances that interfere with the normal functions of the ER, such as glucose deprivation, aberrant ER Ca²⁺ level, and viral infection, cause accumulation of misfolded/unfolded proteins in the ER. When the abundance of misfolded/unfolded proteins exceeds the capacity of the ER’s protein folding machinery, the unfolded protein response (UPR) will be triggered via activation of three ER transmembrane proteins: pancreatic ER kinase-like ER kinase (PERK), inositol-requiring enzyme 1 α (IRE1α), and activating transcription factor 6 (ATF6). Under normal condition, these proteins are inactivated by binding to the resident chaperon molecule GRP78 (glucose-regulated protein 78 kDa) located in the lumen of the ER. Upon ER stress, GRP78 is released, leading to the activation of the UPR [54]. This activation alleviates ER stress by reducing the rate of protein synthesis, degrading the unfolded/misfolded proteins through ER-associated protein degradation (ERAD), and producing molecular chaperons that increase the protein folding capacity of the ER. If the UPR is insufficient in relieving ER stress, prolonged time in this state may ultimately result in apoptosis [55].

MANF is neuroprotective by alleviating ER stress-induced cell damage in multiple tissues and disease models [7, 56-59]. We have reported that MANF expression in the brain was upregulated by alcohol exposure during development [43] and MANF deficiency caused neurons to become more sensitive to alcohol-induced neurodegeneration [60]. In this review, we will discuss the role of MANF in neurodevelopment and its potential as a trophic factor to ameliorate alcohol-induced ER stress and neurodegeneration.

The Discovery of MANF

MANF Protein Structural Domains and Predicted Functions

MANF was first discovered as a neurotrophic factor secreted by the rat mesencephalic type-1 astrocyte cell line [4]. It was purified from the conditioned media and discovered to be selectively neuroprotective to cultured embryonic dopaminergic neurons [4]. It was also found to be homologous to a large portion of the predicted human arginine-rich protein (ARP), missing only the ARP N-terminal arginine-rich sequence [4, 61]. The region of the human ARP gene ARMET (arginine-rich mutated in early-stage tumors), responsible for coding the arginine-rich sequence, was often mutated in different types of cancer. Sequence variations in this region were also found as normal polymorphisms [61, 62]; however, it was later discovered to be located in the 5’ untranslated region (UTR) and not translated in vivo [4]. As a result, human APR was renamed as MANF. The human MANF gene contains 4 exons and is located on the third chromosome.

Human MANF is 18 kDa and contains 179 amino acids, including a signal peptide of the first 21 amino acids in the N-terminus. This peptide targets the nascent MANF protein for the ER in which the peptide is cleaved before MANF maturation [4, 8]. The crystal structure of human MANF is composed of two globular alpha helical domains connected by a flexible linker [13, 63-65]. MANF orthologs have been characterized in vertebrates including the rat (Rattus norvegicus), mouse (Mus musculus), and zebrafish (Danio rerio), as well as in invertebrates such as the fruit fly (Drosophila melanogaster), nematode (Caenorhabditis elegans), aphid (Acyrthosiphon pisum), and sea sponge (Suberites domuncula) [4, 8, 17, 18, 66-68]. MANF is an evolutionary conserved protein [69]. Human and mouse MANF share 98% amino acid identity, while human and Drosophila MANF are 48% identical. Its function is conserved among different species as the expression of recombinant human (rh) MANF was able to rescue the phenotypes in multiple vertebrates and invertebrate MANF-deficient models [66, 70, 71]. Except for CDNF, MANF shows little sequence similarity with any other neurotrophic factors, making it a challenge to predict its function. Accordingly, most proposed mechanisms regarding the function of MANF are based on its protein domains that are similar to other proteins with known function (Table 1).

Table 1.

MANF protein structural domains and predicted functions

Locations	Domains	Predicted functions	References
N-terminus	Saposin-like protein (SAPLIP) domain	Interacts with cell membrane lipids and mediates extracellular MANF intake	[65, 66]
C-terminus	SAP (SAF-A/B, Acinus and PIAS)-like domain	Anti-apoptosis	[13]
		Binds to NF-kB p65 subunit and inhibits NF-kB mediated transcriptional activation of target genes	[72, 73]
		Inhibits ADP release and ATP binding to GRP78, thus inhibits substrate protein release and stabilizes GRP78-substrate protein complex	[74]
C-terminus	CXXC motif	Enhances protein folding	[69]
		Cytoprotection	[70, 75, 76]
C-terminus	RTDL sequence	ER retention	[9, 77, 78]

The N-terminal of MANF contains a saposin-like protein (SAPLIP) domain (Table 1). SAPLIP is known to mediate interactions with the cell membrane and free lipids [79]. As a result, MANF is predicted to interact with lipids on cell membranes and it is proposed that cell membrane lipids may mediate extracellular MANF intake [65]. Recently, C. elegans and human MANF were shown to bind to extracellular lipid sulfatide, promoting internalization of extracellular MANF [66].

The C-terminal of MANF contains an SAP (SAF-A/B, Acinus and PIAS)-like domain which is homologous to the SAP domain of Ku autoantigen p70 subunit (Ku70) [13] (Table 1). Ku70 is an anti-apoptotic protein, inhibiting apoptosis by interacting with and inactivating Bax protein [80]. MANF, particularly the C-terminus of MANF, was shown to be as efficient as Ku70 in protecting neurons intracellularly against Bax-dependent apoptosis [13]. In addition, inflammation causes MANF to translocate into the nucleus and bind with the NF-kB p65 subunit through the SAP-like domain. This interferes the binding of NF-kB with its target genes and inhibits NF-kB mediated transcriptional activation of these genes [72, 73]. Recently, MANF was reported as a nucleotide exchange inhibitor due to its selective interaction with the nucleotide binding domain of ADP-bound GRP78 through its SAP-like domain [74]. The binding of MANF inhibits both ADP release and ATP binding to GRP78, thus inhibiting substrate protein release and stabilizing the GRP78-substrate protein complex (Yan et al. 2019).

MANF has eight conserved cysteine residues across all species including two CXXC motifs (one in the N-terminus, one in the C-terminus), which have been found in many protein disulfide isomerases (PDIs) [65, 81] (Table 1). The disulfide bridge, formed by MANF C-terminus CXXC motif, may facilitate the clearance of misfolded proteins from the ER by enhancing protein folding through its predicted disulfide isomerase activity [69]. It has been shown to be an essential part of the protective effect of MANF. Mutation of the C-terminus CXXC motif in Drosophila MANF (DmManf) destroyed the in vivo cryoprotection function of full-length DmManf [70]. It was also required for the neuroprotective activity of MANF in mice primary superior cervical ganglion (SCG) neurons [75]. In addition, MANF CXXC tetrapeptide added to culture media protected a human T lymphocyte cell line from death receptor Fas-induced apoptosis [76].

At the very end of the C-terminus, MANF has a RTDL sequence that resembles the canonical ER retention sequence KDEL [9, 77] (Table 1). KDEL and KDEL-like sequences in a protein can be recognized by KDEL receptors (KDELRs) that prevent secretion and facilitate protein retrieval back into the ER. Although MANF can be secreted under pathological conditions, it is mostly retained in the lumen of the ER. MANF is retained via KDEL receptors located in the Golgi apparatus and on the cell surface. Deletion of the RTDL sequence from MANF resulted in re-localization of MANF to the Golgi apparatus and increased MANF secretion [7, 77, 78].

The specific cytoplasmic receptor for MANF has not been identified. Recently, the neuronal and synapse-enriched immunoglobulin superfamily member neuroplastin (NPTN) is suggested as a cell membrane receptor for MANF, mediating MANF suppression of NF-kB activation [82]. In addition, new evidence has shown that MANF is able to interact with all three ER transmembrane sensors including PERK, IRE1α, and ATF6, with the highest affinity observed in the MANF-IRE1α interaction [83]. By binding to IRE1α and decreasing its oligomerization and phosphorylation, MANF attenuates UPR and promotes neuronal cell survival [83].

MANF Expression in Adult Tissues

MANF is widely expressed in Drosophila, zebrafish, rodents, and humans [14, 15, 17, 18]. MANF was universally expressed with high expression level in tissues with active secretory and metabolic functions such as the liver, pancreas, testis, salivary glands, and brain regions responsible for energy homeostasis and hormone production (hypothalamus and pituitary gland) [14, 16, 84]. Relatively low expression was detected in the lung, skeletal muscle, kidney, heart, and spleen [14]. Like the expression pattern in rodent, MANF was also present in neuronal and non-neuronal tissues in humans [14, 85].

In the adult rodent brain, MANF mRNA and protein are both detected at high levels in neurons of various regions. In the cerebral cortex, strong MANF expression was observed in cortical layers II–VI. In the hippocampus, MANF was detected in the CA1–CA3 pyramidal neurons and in the granule cell layer of dentate gyrus. In the thalamus and hypothalamus, MANF was detected in the anterodorsal thalamic nucleus, the supraoptic nucleus, and the tuberomam-millary nucleus. MANF protein was also expressed in the basal ganglia including the striatum and the substantia nigra. Notably, Purkinje cells in the cerebellum show especially strong MANF expression, while the mitral cell layer of the olfactory bulb also exhibits MANF expression [14, 15].

MANF in Neurodevelopment

Developmental Expression of MANF in the CNS

Although MANF is detected in most regions of the adult brain, its expression in the CNS begins earlier and is stronger during development. MANF protein was detected in the mouse brain at embryonic day E9.5 spreading regions of the forebrain, midbrain, and hindbrain. During E12.5–E17, when many of the CNS neurons begin to differentiate, MANF expression was observed in the neocortex, median sulcus, striatum, hypothalamus, substantia nigra, and spinal cord [14, 16]. Rodents’ CNS development continued in postnatal days. Strong MANF expression extended to the postnatal rodent CNS. Analysis in both mouse and rat revealed that MANF was expressed in the mitral cell layer of the olfactory bulb, cerebral cortex, hippocampus, thalamus, hypothalamus, and substantia nigra during postnatal day PD 1–PD 30 [14, 15].

Studies in zebrafish and Drosophila indicated that Manf was a maternal contributed gene with strong expression in the embryos shortly after birth. Similar to rodents, manf was also widely expressed in the developing zebrafish neurons in the forebrain, optic tectum, basal ganglia, thalamus and hypothalamus, and cerebellum [17]. Interestingly, Drosophila DmManf was mainly expressed in astrocyte-like glial cells surrounded by dopaminergic neurons in the ventral nerve cord at larval stages [18], but it was detected in dopaminergic neurons in the adult brain [86].

Role of MANF in Neurodevelopment

Neurodevelopmental delay and defects have been reported in human patients with MANF mutations. The first human patient with MANF mutation was identified in an exome sequencing of a cohort of 150 patients with various Mendelian neurocognitive disorders. A homozygous MANF splice site mutation was identified in a 22-year-old female who dealt with type 2 diabetes, obesity, hypothyroidism, primary hypogonadism, short stature, alopecia, bilateral sensorineural deafness, myopia, microcephaly, and mild intellectual disability [87]. Recently, whole-genome sequencing for 32 children with syndromic diabetes further identified a 17-year-old female with a homozygous frameshift variant in MANF. She has multisystem disorders characterized by childhood-onset of diabetes, short stature, and neurodevelopmental defects including bilateral sensorineural deafness, microcephaly, and developmental delay [88].

MANF deficient animal models listed in Table 2 demonstrate that MANF regulates the development of neurons. Global MANF knock-out (KO) mice showed abnormal cerebral cortex development with altered cerebral cortex thickness and cell density; however, there was no significant difference in the number of neural stem cells (NSCs), cell proliferation, neurogenesis, and the rate of programmed cell death [20, 71]. Examination of the distribution of bromod-eoxyuridine (BrdU) and several neuronal specific markers further indicated that the abnormal cerebral cortex of developing MANF KO mice was due to the disruption of neuron migration and that MANF was essential for the migration of neurons within the embryonic cerebral cortex [20]. Interestingly, neuron-specific MANF KO showed increased neurogenesis in both the developing and mature mouse brain [please add the additional citation in Table 2 also in here]. In addition, MANF^−/− neurons exhibited decreased neurite extension in both in vitro differentiated NSCs and a retinoic acid-induced mouse neuronal cell line, as well as in the developing cortex in vivo [19, 20]. MANF deficiency resulted in ER stress and impaired protein synthesis, as well as hypoactivation of Akt/mTOR and Erk/mTOR signaling pathways [19, 20, 89]. These may be the underlying mechanisms causing defects in neurite extension.

Table 2.

MANF deficiency interrupts neurodevelopment

Subjects	Types of deficiency	Ages	Phenotypes	References
C. elegans	manf-1 mutation	Adult (day 3–9)	Viable and healthy, slower growth rate, normal neuronal development, degeneration of dopaminergic neurons in adult, increased systemic ER stress	[66, 90]
Drosophila	Zygotic DmManf mutation	1st instar	Lethal at 1st instar, loss of dopaminergic neurites and reduced dopamine	[18]
	Maternal zygotic DmManf mutation	Embryonic stage 16	Lethal at embryonic stage, loss of all TH-positive neurites and nonapoptotic neuron death	[18]
Zebrafish	Morpholino manf knockdown	3 days post fertilization	Decrease of th1- and th2-expressing dopaminergic neurons, reduced dopamine level, upregulation of pax2a and nr4a2b	[17]
Mouse	Global Manf knockout	E15.5-P7	Slower neuron migration, decreased neurite extension, altered cerebral cortex thickness and cell density, activated UPR and decreased protein synthesis	[20]
	Neuron-specific Manf knockout	E13.5-P14, adult, aging	Activated UPR, increased neurogenesis	[89, 91]
Human	Homozygous frameshift variant in MANF	17 years old	Childhood-onset of diabetes, short stature, bilateral sensorineural deafness, microcephaly, and developmental delay	[88]
	Homozygous MANF splice site mutation	22 years old	Childhood-onset of diabetes, obesity, short stature, hypothyroidism, primary hypogonadism, alopecia, myopia, bilateral sensorineural deafness, microcephaly, and mild intellectual disability	[87]

MANF and ER stress

ER Stress and MANF Expression and Secretion

ER stress induced by ischemia or chemical treatments with tunicamycin or thapsigargin cause upregulation of MANF expression in neurons. Middle cerebral artery occlusion (MCAO) rat model of cerebral ischemia induced ER stress and MANF expression in both neurons and glial cells in the ischemic region [11, 56, 92-94]. Tunicamycin treatment upregulated MANF expression in the human neuronal cell line SH-SY5Y and primary cultured rodent neurons [56, 93]. Exposure to tunicamycin also significantly increased the expression of MANF in the brain of neonatal mice [95].

Most ER stress responsive genes such as GRP78, GRP94, and calreticulin contain an ER stress response element (ERSE) in their promoter region, with the consensus sequence of CCAAT-N(9)-CCACG [96, 97]. ERSE is also present in the promoter region of MANF. In 2001, a new ER stress response element II (ERSE-II) was discovered in the promoter region of human HERP (homocysteine-induced endoplasmic reticulum protein) gene, with the consensus sequence of ATTGG-N-CCACG [98]. Manf was the second gene found that has ERSE-II in its promoter region [8]. In vitro studies have shown that Manf ERSE and ERSE-II in the promoter were recognized and regulated by UPR molecules ATF6 and spliced X-box binding protein 1 (XBP1s) [98-101]. MANF expression was reduced in cells lacking both ATF6 and XBP1s [102]. Decreased association of XBP1s to MANF promoters was reported in a mouse model of spinocerebellar ataxia, resulting in reduced MANF expression and degeneration of Purkinje cells [103]. On the contrary, MANF was upregulated in mouse cardiomyocytes in which ATF6 was constitutively expressed [104].

Intracellular trafficking and secretion of MANF is also regulated by ER stress. Increased MANF secretion was associated with several ER stress condition associated diseases, including kidney disease in rodent models and diabetes in human [105-108]. Under non-stressed conditions, MANF was only minimally secreted with a significant proportion being retained in the ER [58]. The RTDL sequence in the C-terminus of MANF functions as an ER retention signal. It binds to KDEL receptors in the Golgi apparatus and cell membrane that are responsible for retaining MANF into the ER and preventing its secretion [9, 77]. Unlike the canonical ER retention signal KDEL, RTDL in MANF has a lower affinity to the KDEL receptors. It has been proposed that under ER stress, when ER stress-response proteins with both KDEL and RTDL are induced and KDEL receptors do not change [109], proteins with KDEL outcompete the ones with RTDL in binding to KDEL receptors [110]. As a result, secretion of proteins with the RTDL sequence, such as MANF, increases [77].

Interestingly, while various ER stressors can induce MANF expression in cells, MANF secretion seems to be tightly regulated by cellular calcium levels. Tunicamycin, thapsigargin, and dithiothreitol are commonly used agents that induce ER stress by inhibiting glycosylation, decreasing ER calcium levels, and altering ER redox status, respectively. A significant amount of MANF was secreted from cardiomyocytes and HeLa cells when treated with thapsigargin; however, not when treated with tunicamycin and dithiothreitol, suggesting that decreased ER calcium may trigger MANF secretion [7]. Additionally, MANF secretion was induced by other ER calcium depletion reagents. The calcium-sensitive MANF secretion was RTDL-independent as thapsigargin increased the secretion of a modified form of MANF with no RTDL. Further evidence indicated that MANF retention in the ER was partially governed by its physical interaction with the ER resident chaperon GRP78 in a calcium-dependent manner [7]. In contrast, recent study using microscale thermophoresis demonstrated that the binding of purified recombinant MANF and GRP78 was not dependent on calcium [111]. MANF and GRP78 are frequently co-upregulated in cerebral ischemia models and genetic interaction of MANF and GRP78 has been reported in Drosophila [94, 112, 113]. Enforced expression of GRP78 attenuated the secretion of MANF and led to its intracellular accumulation [9].

Role of MANF in ER Stress and Neurodegeneration

MANF^−/− neurons are more susceptible to ER stress-induced neurodegeneration. For example, adult C. elegans with manf-1 mutation exhibited increased ER stress and dopaminergic neuron degeneration [90, 114]. Conditional MANF KO in the epithelial cells and neurons of the mouse inner ear resulted in UPR activation with progressive outer hair cell loss in the cochlea [115]. NSCs isolated from constitutive MANF KO mice at embryonic day E13.5 showed increased UPR activation upon in vitro differentiation [20]. CNS-specific MANF KO demonstrated that MANF deficiency results in an increased expression of UPR genes from all three UPR pathways in the brain of embryos, newborns, and adults [89]. In the MANF^−/− aging brain (16 months old), all three UPR pathways were activated in the substantia nigra [89]. MANF deficient cortical neurons isolated from these mice were more vulnerable to thapsigargin-induced ER stress and cell death in vitro [89].

Elevated ER stress is commonly observed in various neurodegenerative diseases and CNS injuries [116]. MANF has been implicated to alleviate ER stress and protect neurons from ER stress-induced neuronal death. The role of MANF in various neurodegenerative disease models including ischemic stroke, PD, AD, spinocerebellar ataxia (SCA), and eye injury is summarized in Table 3.

Table 3.

MANF in neurodegenerative diseases and neuronal injuries

Subjects	Models	Discoveries	References
Ischemic stroke
Rat	CCAO	Global forebrain ischemia caused widespread MANF expression in the hippocampus and cerebral cortex.	[14]
Rat	MCAO	MANF expression was upregulated in ischemic cortical neurons and glial cells, which may be protective to the ischemic brain.	[56, 93, 94]
Rat	MCAO	Intracerebral injection of rhMANF or AAV-MANF significantly reduced the volume of cerebral infarction and attenuated neuronal apoptosis.	[11, 92]
Rat	MCAO	Intracerebroventricular injection of rhMANF protected neurons from ischemia-induced apoptosis and attenuated the elevation of GRP78 and XBP1s.	[117]
Rat	MCAO	MRI indicated that intracerebroventricular injection of MANF at early stage of ischemia improved neurological function, reduced infarct volume, and alleviated brain injury.	[118]
Rat	MCAO	Poststroke administration of MANF promoted functional recovery and increased the number of phagocytic immune cells in the ischemic brain.	[119]
Rat	MCAO	Poststroke brain parenchyma injection of AAV-MANF in the peri-infarct region increased the expression of immune related genes and downregulated phagocyte proteins S100A8 and S100A9.	[120]
Rat	MCAO	Intracerebroventricular delivery of MANF improved poststroke neurobehavioral recovery by increasing total vessel surface area, increasing the amount of microvessel branch points, and activating the vascular endothelial growth factor pathway.	[121]
Rat	MCAO	Bone marrow mesenchymal stem cells (BMSC)-secreted MANF can induce M1 to M2 shifting of the microglia/macrophages, which contributed to BMSCs-induced brain repair in ischemic stroke.	[122]
Rat	MCAO	DHA can induce MANF expression in neurons and astrocytes, which reduced infarct size and improved neurological function after ischemic stroke.	[123]
Parkinson’s disease (PD)
Rat	6-OHDA	Intrastriatally injected MANF protected nigrostriatal dopaminergic neurons from 6-OHDA induced degeneration and restored nigrostriatal dopaminergic system neuronal function.	[10]
Rat	6-OHDA	Combined nigral overexpression of both MANF and CDNF synergistically protected dopaminergic neurons.	[124]
SH-SY5Y cells	6-OHDA or α-synuclein	Addition of MANF protein in culture media attenuated 6-OHDA or α-synuclein induced apoptosis via upregulation of GRP78.	[113]
Rat and SH-SY5Y cells	6-OHDA	Intrastriatal injection of AAV9-MANF promoted nigral dopaminergic neuron survival and long-term behavior improvement. MANF inhibited 6-OHDA induced ER stress and activated PI3K/Akt/mTOR pathway in vitro.	[125]
SH-SY5Y cells	6-OHDA	MANF protected against 6-OHDA induced apoptosis in SH-SY5Y cells via upregulating HSP70.	[126]
SH-SY5Y cells	6-OHDA	MANF protected against 6-OHDA induced apoptosis in SH-SY5Y cells via inhibiting autophagy through ROS-AMPK/mTOR signaling pathway.	[127]
SH-SY5Y cells	6-OHDA	MANF protected against 6-OHDA induced cytotoxicity by potentiating the Nrf2-related survival mechanism through the PI3K/Akt/GSK3β pathway.	[128]
Mouse and SH-SY5Y cells	MPTP/MPP+	MANF attenuated neuronal lesion in MPTP/MPP+-induced PD mice with improved mitochondrial function and reduced oxidative stress.	[129]
C. elegans	α-Synuclein	Dopaminergic neuron specific MANF overexpression alleviated progressive neuronal degeneration and prevented locomotion defects by regulating ER stress and autophagy pathways.	[130]
Human	PD patients	Circulating MANF concentrations in serum were significantly higher in PD patients than controls.	[131]
Human	PD patients	MANF expression was not changed in the hippocampus of PD patients.	[132]
Alzheimer’s disease (AD)
Mouse	APP/PS1 double transgenic	The expression of MANF, GRP78, and CHOP was increased in the cortex and hippocampus of APP/PS1 mice.	[12]
Cell culture	Aβ1–42 peptide	MANF attenuated Aβ-induced ER stress and cell death, whereas MANF knockdown activated UPR and aggravated Aβ neurotoxicity.	[12]
Human	Pre-AD and AD patients	The number of MANF-expressing neurons was significantly increased in the inferior temporal gyrus of the cortex (ITGC) of pre-AD and AD patient brains.	[133]
Spinocerebellar ataxia (SCA)
Mouse	SCA17 knock-in	MANF expression was decreased in mutant Purkinje cells. MANF overexpression ameliorated mutant TBP-mediated Purkinje cell degeneration.	[103]
Mouse	SCA17 knock-in	Piperine antagonized ER stress by activating MANF expression in vitro and ameliorated SCA17 neuropathology in mouse brain.	[134]
Eye injury
Drosophila and mouse	Light damage and transgenic	MANF was overexpressed in innate immune cells in damaged fly and mouse retina, which promoted alternative (M2) activation of innate immune cells and enhanced neuroprotection and tissue repair.	[135]
Rat	Hypoxia and COHT	Intravitreal injection of MANF protected retinal ganglion cell from hypoxia-induced ER stress and apoptosis in both in vitro and in vivo models.	[136]
Rat	S334ter-3 transgenic	Intravitreal injection of rhMANF protected photoreceptors from degeneration in transgenic rats carrying a murine rhodopsin mutant S334ter-3.	[137]
Retinal cell culture and explants	Tunicamycin	Addition of rhMANF protected rat retinal precursor R28 cells and ex vivo cultured mouse retinal explants from tunicamycin-induced ER stress and cell death.	[138]
Mouse	Streptozotocin-induced diabetic keratopathy (DK)	Subconjunctival injection of MANF inhibited hyperglycemia-induced ER stress and promoted corneal epithelial wound healing and nerve regeneration.	[139]
Mouse	Aging and light damage	MANF supplementation reduced light damage and increased integration after retinal cell transplantation to the aged retina by immune modulation.	[140]

Abbreviations: CCAO, bilateral common carotid arteries occlusion; MCAO, middle cerebral artery occlusion; 6-OHDA, 6-hydroxydopamine; MPTP/MPP+, neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its metabolite 1-methyl4-phenylpyridinium ion; APP/PS1, amyloid precursor protein/presenilin1; SCA17, spinocerebellar ataxia 17; TBP, TATA box binding protein; COHT, chronic ocular hypertension

Ischemic stroke can induce ER stress in neurons through depletion of ER calcium, aggregation of proteins, and accumulation of lipid peroxidation products [141]. MANF expression was induced in the ischemic cerebral cortex for both neurons and glial cells in rodent cerebral ischemia models [56, 93, 94]. Pretreatment of rhMANF or MANF overexpression by adeno-associated virus (AAV) significantly reduced the infarction area and amount of neuron death in the ischemic brain [11, 75, 92, 117, 118]. Poststroke administration of MANF promoted functional recovery [119], modulated immune cell-related gene expression and recruited phagocytic immune cells in the peri-infarct brain regions [120], promoted the migration of neuroblast cells towards the infarcted area [142], and facilitated poststroke cerebral angiogenesis [121]. Furthermore, MANF was responsible for bone marrow mesenchymal stem cell (BMSC)-mediated microglia shifting from proinflammatory M1 state to anti-inflammatory M2 state, which contributes to BMSCs-induced postischemic brain repair [122]. Chemicals or nutritional supplements that can induce MANF expression in the brain may serve as a MANF-based therapeutic agent for ER stress-related neurological diseases. Recently, MANF expression in the rodent ischemic brain was reported to be induced by DHA supplementation that enhances neurogenesis, reduces infarct size, and improves neurological function after experimental ischemic stroke [123].

MANF has been implicated in the neuroprotection of Parkinson’s disease (PD) and Alzheimer’s disease (AD) where ER stress and UPR are activated. PD is a progressive neurodegenerative disorder characterized by extensive dopaminergic neuron degeneration in the substantia nigra. Elevated ER stress and UPR activation are often observed in toxicological models resembling sporadic PD and in the post-mortem human PD brain [143, 144]. Increased MANF protein level was found in the serum of human PD patients, but not in the hippocampus of the brain [131, 132]. Cell and animal models have been used to show that MANF was neuroprotective and neurorestorative in models of PD (reviewed by [1, 145, 146]). In rodent PD models, intrastriatal delivery of MANF protect and restored the function of dopaminergic neurons from 6-hydroxydopamine (6-OHDA)-induced degeneration [10, 124, 129]. In α-synuclein-induced C. elegans model of PD, MANF overexpression in the dopaminergic neurons alleviated neurodegeneration by regulating ER stress and autophagy [130]. In vitro studies using SH-SY5Y cells further indicated that intracellular overexpression of MANF alleviated 6-OHDA-induced ER stress and decreased expression of UPR genes p-eIF2α, ATF4, CHOP, XBP1s, and ATF6. Extracellular addition of rhMANF protected cells through the activation of PI3K/AKT pathway, the inhibition of autophagy, and the induction of ER chaperon GRP78 and heat shock protein 70 (HSP70) [113, 125-128].

Extracellular accumulation of amyloid β-peptide (Aβ) is one of the pathological hallmarks of Alzheimer’s disease (AD) [147, 148]. Continuous accumulation of Aβ induces ER stress in neurons, contributing to synapse dysfunction and neurodegeneration characteristic of AD [149]. The number of MANF-expressing neurons was significantly increased in the inferior temporal gyrus of the cortex (ITGC) of pre-AD and AD patient brains, suggesting the potential use of MANF as a diagnostic biomarker for early stage AD [133]. MANF upregulation observed in PD and AD patients is in line with the increased ER stress and activated UPR associated with PD and AD pathogenesis. It is postulated that calcium depletion and chronic ER stress lead to the upregulation of MANF in AD patient brains. The induced MANF may exert neuroprotective roles in the neurodegenerative brain through attenuating ER stress. Amyloid precursor protein/presenilin 1 (APP/PS1) double transgenic mice are widely used as an animal model for AD by exhibiting remarkable β-amyloid accumulation in the brain [150]. Increased expression of MANF and ER stress markers GRP78 and CHOP were found in the cortex and hippocampus of APP/PS1 mice [12]. In vitro analysis using SH-SY5Y and N2a neuronal cell lines revealed that MANF overexpression ameliorated Aβ-induced ER stress and overall cell death, whereas MANF knockdown exacerbated UPR and aggravated Aβ neurotoxicity [12].

Spinocerebellar ataxia 17 (SCA17) is a cerebellar neurodegenerative disease caused by polyglutamine (polyQ) expansion in TATA box binding protein (TBP), leading to TBP misfolding and the degeneration of cerebellar Purkinje cells. In an inducible SCA17 knock-in mouse model, mutant TBP levels were higher in older mice likely due to age-related reduction of the chaperone activity of heat shock cognate 71 kDa protein (HSC70), resulting in impaired cellular capacity to handle misfolded proteins and ER stress in aged mice [103]. Mutant TBP reduced the association of XBP1s with MANF promoters, resulting in decreased MANF expression and degeneration of Purkinje cells [103]. Overexpression of MANF ameliorated mutant TBP-mediated Purkinje cell degeneration and ER stress in SCA17 knock-in mice [103, 134]. Several MANF inducing chemicals, including piperine, genistein, glyburide, formononetin, and pinosylvin, were identified in a large-scale screening of FDA-approved chemicals [134]. Further tests confirmed that by inducing MANF overexpression, piperine can suppress tunicamycin- or mutant TBP-induced ER stress in cell culture and ameliorate SCA17 neuropathology in the mouse brain [134].

MANF was also reported to regulate and restore neuronal function in injured eyes. High levels of MANF are expressed in both the invertebrate and vertebrate eyes [60, 137, 151, 152]. MANF exerts a protective role in multiple eye injury models including light damage and rhodopsin mutation induced photoreceptor degeneration [135, 137]. Several studies support the role of MANF in protecting retinal cells from ER stress-induced damages. In rodent models, MANF protect retinal cells from hypoxia-, hyperglycemia-, or tunicamycin-induced ER stress and apoptosis in both in vitro and in vivo models [136, 138, 139]. Additional mechanism is dependent on its modulation of immune cells. In both Drosophila and mice eyes, intensive light exposure stimulated MANF expression in innate immune cells, which was both necessary and sufficient to promote retinal repair [135, 153]. In addition, changes in the immune environment of aged eyes limited the retinal homeostatic capacity, causing more photoreceptor death after light damage, which can be reversed by MANF supplementation [140]. MANF also increased the integration efficiency of transplanted cells into the aged retina, while the beneficial effect was blocked in immune-deficient mice carrying MANF-irresponsive macrophages [140].

MANF as a Neurotrophic Factor Protecting Against Alcohol-Induced Neuronal Damages in the Developing CNS

Alcohol-Induced Damages to the Developing Brain and Neurobehavioral Deficits

Alcohol is a teratogen. It was first reported by Lemoine in 1968 that alcohol potentially posed adverse effects to children of alcoholic parents, including facial anomalies, growth deficiencies, and behavioral and cognitive impairments [154]. In 1973, Jones and Smith reported more cases exhibiting similar abnormalities and assigned them under the label “fetal alcohol syndrome” (FAS) [155]. Now, it is clearly known that prenatal alcohol exposure (PAE) impacts the development of the fetus and can give rise to a wide variety of physiological, cognitive, behavioral, and neurological abnormalities, which are collectively known as fetal alcohol spectrum disorders (FASD). FAS defines the most severe end of the spectrum. Among the various organ systems, the nervous system is the most critically affected system by PAE [156]. Alcohol exerts detrimental effects on the neuroanatomical measurements of the developing brain [157]. The most well characterized neuroanatomical change is the reduction of brain volume demonstrated in both human patients and animal models [158-160]. Early human brain autopsy studies of severe PAE children revealed microcephaly, incomplete development of the cerebral cortex, malformation, and agenesis of the corpus callosum, shrinkage of cerebellum, enlargement of the ventricles, and neuroglial heterotopia [155, 161-163]. In the last three decades, magnetic resonance imaging (MRI) has enabled the detection of subtle brain malformations in almost every brain region, for a larger population of PAE individuals, including those with less extreme cases. Microcephaly and volume reduction in specific brain regions including the frontal lobe, hippocampus, basal ganglia, thalamus, and cerebellar vermis were frequently reported in children with PAE (reviewed in [164-166]). Differences in the thickness of the cerebral cortex in the frontal lobe, increased gray-matter density in the parietal lobe, and reduced white-matter integrity were all reported in children with FASD [167-169]. In addition, abnormalities have often been found in the corpus callosum and increased variability in the shape of the corpus callosum was suggested to serve as potential diagnostic criteria for FAS and related brain disorders [170, 171]. Volume reduction and displacement of the anterior cerebellum vermis was also reported in prenatal alcohol exposed children and adolescents [172-174].

The structural changes of the brain are responsible for the subsequent cognitive and behavioral outcomes observed in prenatal alcohol exposed individuals (see [175, 176] for comprehensive reviews). A meta-analysis showed that PAE was associated with many neurobehavioral deficits in human [177]. Decreased brain volume correlates with deficits in cognitive function and FASD children had significant reduction in some regions of the basal ganglia and diencephalon as well as lower IQ scores [178]. Significantly reduced cortical gyrification across the cerebral cortex was observed in a large sample of children with PAE compared to controls, which was correlated with lower IQ scores [179]. Cerebellum controls motor activities such as balance and coordination along with cognition and emotion [180]. Structural abnormalities in the cerebellum vermis may be responsible for motor and cognitive deficits seen in individuals with FASD [181-183]. Abnormalities of the corpus callosum were associated with deficits in attention, verbal learning, and executive function of prenatal alcohol exposed individuals [184-187].

Rodent models of FASD have provided important insights into the effects of dosage, duration, and timing of alcohol exposure on brain development and behavioral outcomes. These models offer better control of variables such as genetic background, health and nutrition status, and stress [188]. Although the human and rodent brains have anatomical and functional differences, they exhibit structural analogy and similar developmental stages. A typical 40-week human pregnancy is divided into three trimesters, with 0 to 13 weeks as the first trimester, 14 to 26 weeks as the second trimester, and 27 to 40 weeks as the third trimester. Human neural tube formation is complete at approximately 4 weeks of gestation, followed by neurogenesis, proliferation, migration, and differentiation of specific regions of the brain throughout the pregnancy. Differentiation and synaptogenesis continue into the postnatal period and through early childhood. The growth rate of human brain peaks at approximately 4 months post-birth as measured by the increase in volume and gradual decrease [189-192]. Rodent pregnancy is much shorter than human pregnancy (Fig. 1). Gestation period in mice is approximately 21 days, with gestation days (GD) 1–10 corresponding to the first trimester in human pregnancy and GD 11–21 as the second trimester equivalent [193]. The third trimester equivalent correlates with postnatal days (PD) 1–10 in mice. Various alcohol administration paradigms for rodents have been used to model alcohol exposure during the first and second trimesters of human pregnancy, including liquid diet, intraperitoneal injection, vapor chamber, voluntary drinking, and oral/intragastric gavage to pregnant dams [194]. Alcohol administration to neonatal pups through oral/intragastric gavage, artificial feeding, vapor chamber, and subcutaneous injection were used to model third trimester alcohol exposure in humans [188].

Fig. 1.

Timeline of mouse brain development and the effects of alcohol exposure. The approximate timing of major neurobiological processes is represented by the shaded horizontal bars. Alcohol administration routes and effects of alcohol are listed for each alcohol sensitive period

Alcohol exposure at any point during pregnancy can cause damage to the function and growth of the brain; however, the fetus brain exhibits varying vulnerability to alcohol at different development stages (Fig. 1). In mouse models, the first alcohol sensitive period is between GD 5 and 10, in which the neural tube is forming along with the initiation of neurogenesis. Alcohol exposure during this period often causes considerable craniofacial malformations and neurological defects with behavioral consequences, which are reminiscent of clinical features of children with FAS [195-198]. The second critical period occurs between GD 11 and 21. During this period, neural stem cells migrate out from germinal zones and give birth to neuroblasts, which migrate and differentiate into neurons and glial cells in specific areas of the brain [199]. Therefore, PAE during this developmental stage particularly affects neuronal proliferation, migration, and differentiation in the cerebral cortex, hippocampus, cerebellum, and basal ganglia [200-205]. The third critical period is the early postnatal days. While the human brain exhibits more prenatal maturation, the rodent brain undergoes substantial development in the postnatal period. Studies focused on the third trimester-equivalent model suggest that rodent brain in the first 2 weeks after birth was susceptible to the neurotoxic effects of alcohol [45, 206, 207]. During this period, the mouse brain undergoes continuous neurogenesis in the cerebellum and the dentate gyrus of the hippocampus. Meanwhile, gliogenesis, myelination, axogenesis, and synapse maturation also occur. The third trimester-equivalent alcohol exposure causes neuronal death [43, 60, 207-212], synaptic disruption [213], and reactive gliosis and myelination impairment [214-216]. It was demonstrated that single alcohol exposure during the synaptogenesis period triggered massive-scale apoptotic neuronal death in several major regions of the developing rat brain [207]. Similar neuronal apoptosis was also observed in the developing mouse [210, 211] and monkey brains [217]. Apoptotic deletion of neurons readily explains, at least in part, the adverse effects of prenatal alcohol exposure in FASD, such as reduction of brain volume, dysgenesis of the corpus callosum, and neurobehavioral disturbances [218].

Alcohol-induced neuronal damage in the mouse brain is associated with profound neurobehavioral deficits similar to that of FASD children (reviewed in [219]). For example, chronic maternal alcohol consumption during pregnancy, maintained throughout lactation until wean, leads to increased anxiety-like behavior in the offspring [220]. Acute binge-like alcohol exposure on a single postnatal day triggers apoptotic neuronal death in brain regions comprising an extended hippocampal circuit, leading to severe impairments in spatial learning and memory in juvenile mice [221].

Alcohol Metabolism in the Brain

It is postulated that alcohol metabolites are responsible for some neuronal damages and neurobehavioral deficits [222, 223]. Liver is the primary organ utilized for alcohol metabolism. Most alcohol is oxidized to acetaldehyde and later acetate, while small amounts of alcohol are metabolized through nonoxidative pathways in reactions with fatty acids and phospholipids [224]. Various metabolic enzymes are involved in alcohol oxidation including alcohol dehydrogenase (ADH) in the cytosol, cytochrome P450 2E1 (CYP2E1) in the smooth ER (microsome), and catalase in the peroxisome. ADH is the main enzyme that converts alcohol to acetaldehyde in the liver. Mammalian ADH has five classes. The three isoforms of class 1 ADH 1A, 1B, and 1C are responsible for most of the acetaldehyde production in the human liver [225, 226]. CYP2E1 and catalase are activated upon acetaldehyde generation in the liver when drinking is chronic, or the blood alcohol concentration (BAC) is high with saturated ADH enzymes activity [227, 228]. Acetaldehyde is then further oxidized to acetate by aldehyde dehydrogenase 1 (ALDH 1) in the cytosol and ALDH 2 in the mitochondria [229].

Due to the high ALDH activity in the endothelial cells and oligodendrocytes in the blood-brain barrier, peripheral acetaldehyde can hardly enter the brain; however, alcohol can easily cross the blood-brain barrier (BBB) and be metabolized within the brain (Fig. 2) [230, 231]. While alcohol metabolism in the liver is well characterized, metabolism in the brain is considerably less understood. Functional activity of ADH in the brain was once thought to be absent, until the presence of some isoforms of ADH was identified in specific brain regions. Class 3 ADH (ADH 3) was found to be expressed in rat, mouse, and human brains, especially in the cerebellum Purkinje cells [232]. However, it appeared to oxidize alcohol poorly (K_M greater than 2.5 M), suggesting that ADH 3 has a low affinity to alcohol [233]. ADH 1 and ADH 4 mRNA were also present in the rodent brain, but their functional activity was only detectable in cerebellum granular cells and Purkinje cells, not in the whole-brain homogenates [234]. The above evidence suggests that ADH may only play a minor role in alcohol metabolism in the brain.

Fig. 2.

Alcohol induces ER stress in the brain. Alcohol is oxidized to acetaldehyde and then acetate in the brain. The conversion of alcohol to acetaldehyde is mainly facilitated by catalase and CYP2E1, while ADH only plays a minor role in this process. Acetaldehyde is then further oxidized into acetate by ALDH. Alcohol metabolism triggers ER stress in the brain through several potential mechanisms, including acetaldehyde adducts, homocysteine toxicity, epigenetic alterations, oxidative stress, and perturbation of the ER redox status and Ca²⁺ homeostasis. Temporary ER stress and UPR activation is protective and restores the homeostasis in neurons, while prolonged ER stress leads to neurodegeneration. Abbreviations: CYP2E1, cytochrome P450 isoform 2E1; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; H₂O₂, hydrogen peroxide; NAD⁺, nicotinamide adenine dinucleotide; NADH, the reduced form of NAD⁺; NADP⁺, nicotinamide adenine dinucleotide phosphate; NADPH, the reduced form of NADP⁺; UPR, unfolded protein response

Catalase and CYP2E1 are the primary alcohol metabolization enzymes in the brain (Fig. 2). Catalase is localized in the peroxisome and its expression was detected in neurons [235, 236]. In conjunction with endogenous hydrogen peroxide, it was shown to oxidize ethanol in the brain [237]. Inhibition of catalase activity effectively reduced acetaldehyde production in the brain and blocked alcohol-induced behavioral changes [228, 230, 231, 238]. CYP2E1 is a variant of cytochrome P450. Expression of CYP2E1 in neurons and glial cells can be induced by alcohol exposure in specific regions of the brain, including the cerebral cortex, thalamus, hippocampus, and cerebellum [239-242]. Inhibition or genetic deficiency of CYP2E1 significantly reduced acetaldehyde production in the rodent brain [243, 244]. Using pharmacological and genetic approaches, Zimatkin and colleagues demonstrated in rodent brain homogenates that catalase and CYP2E1 account for about 60% and 20% of alcohol metabolism in the brain in vitro, respectively, while ADH only plays a minor role, if any [245]. Alcohol can enter the fetus by dispersing through the placenta. The developing fetus metabolizes alcohol in the same way as adults, but with reduced efficiency due to the immature alcohol metabolism systems [246]. As a result, the elimination of alcohol in the fetus is slow and largely dependent on maternal alcohol metabolization, posing a critical threat to the developing brain.

ER Stress and Alcohol-Induced Neuronal Damage in the Developing CNS

There are several potential mechanisms for alcohol-induced damages to the developing brain [247], including alterations in neurotransmitters [40], increased oxidative stress and mitochondria damages [248], activation of glial cells and neuroinflammation [214], autophagy [249], disruption of neurotrophic factor functions [42, 158], changes in gene expression and epigenetic modifications [250], and the activation of ER stress [46]. This review will mainly discuss the contribution of ER stress to alcohol-induced neurotoxicity.

It has been well established that ER stress contributes in alcohol-induced damages to multiple major organs including the liver, the pancreas, the heart, and the brain [251]. In a model of alcohol-induced liver injury, ER stress was developed in alcohol-fed mouse liver as indicated by increased expression of GRP78, GRP94, and CHOP [252]. The pancreas is also a vulnerable target for alcohol-induced ER stress. Chronic alcohol feeding caused increased pancreatic levels of XBP1 [253]. The combination of alcohol feeding and XBP1 deficiency resulted in reduced ER function and an upregulation of proapoptotic signals in the pancreas [253-255]. In vitro study indicated that alcohol exposure induces UPR gene expression in pancreatic β-cell lines, isolated murine islets [256] and pancreatic acinar cells [257, 258]. In addition, chronic alcohol exposure induced myocardial ER stress in mice, indicated by increased GRP78 and CHOP, as well as elevated phosphorylation of IRE1α and eIF2α [259, 260]. 4-PBA (4-phenylbutyric acid), a chemical chaperone that inhibits ER stress by decreasing accumulation of misfolded proteins in the ER, was able to ameliorate alcohol-induced cardiomyocyte injury and relieve ER stress in both a H9c2 rat cardiac cell line and rat primary cardiomyocytes [261].

There are several potential mechanisms for alcohol-induced ER stress in neurons, with evidence found in both human and animal studies focused on varying organs such as the liver, heart, muscle, and brain [46]. Possible reasons include the formation of harmful acetaldehyde and protein adducts, disturbance of methionine metabolism causing homocysteine toxicity and altered DNA methylation, production of highly reactive oxygen species, perturbation of oxidative protein folding and the redox status of the ER, and disruption of ER Ca²⁺ homeostasis (Fig. 2). For comprehensive reviews, please refer to [46, 251, 262, 263].

We demonstrated that ER stress played an important role in alcohol-induced neuronal damage in the developing brain and in vitro. Early studies using human SH-SY5Y neuroblastoma cells and cultured rat primary cerebellar granule neurons showed that alcohol alone had little effect on the expression of UPR genes. Even so, alcohol significantly potentiated tunicamycin- and thapsigargin-induced expression of GRP78, CHOP, ATF4, ATF6, and phosphorylated PERK and eIF2α in these neuronal cells, indicating that alcohol was synergistic with tunicamycin and thapsigargin in enhancing ER stress [264]. CHOP is a proapoptotic transcription factor that serves as a key mediator for ER stress-induced cell death [265]. It has been shown that alcohol-induced hepatocellular apoptosis was eliminated in CHOP knock-out mice [266]. Using CHOP^−/− mouse embryonic fibroblasts (MEFs) culture and CHOP siRNA, we showed that CHOP deficiency mitigated alcohol-induced neuronal cell death in vitro [264]. Using a third-trimester equivalent mouse model, we demonstrated for the first time that alcohol exposure induced ER stress in the developing brain [43]. When pups of PD 7 were acutely exposed with alcohol by subcutaneous injection, significant upregulation of UPR genes was induced as early as 4 hours (h) after alcohol exposure and sustained for over 24 h [43]. Surprisingly, unlike the in vitro result, CHOP^−/− and CHOP^+/+ mice exhibit similar susceptibility to alcohol-induced neuroapoptosis, indicating that CHOP knock-out does not offer protection against alcohol-induced damage to the developing brain in vivo [43]. UPR induced activation of IRE1/JNK/SAPK signaling pathway and its effect on Bcl2/Bax expression is also important for ER stress mediated apoptosis [267, 268]. Several Bcl2 family proteins including Bcl2l1, Bak1, Bad, Bid, and Bnip3 have been differentially expressed in the developmental rodent brains in alcohol sensitive and resistant periods [45].

It is known that there is a temporal and spatial susceptibility to alcohol during development. We compared alcohol-induced neuroapoptosis in the brain of mouse pups at PD 4 and PD 12. While alcohol caused wide-spread neurodegeneration in the brain of PD 4 pups (Fig. 3a), little effect was observed in the brain of PD 12 mice [45]. Gene expression profile analyzed by microarray indicated that the expression of proapoptotic genes in the brain was much higher in PD 4 mice than PD 12 mice; conversely, the expression of UPR genes including GRP78, IRE1α, PERK, and activated ATF6 in the brain of PD 12 mice was significantly higher than PD 4 mice [45]. Consistent with this result, we showed that tunicamycin, a more specific ER stress inducer, caused more neurodegeneration in the immature brain [95]. Furthermore, we demonstrated that inhibition of ER stress could protect the alcohol-sensitive immature brain from alcohol-induced neuronal damage. In PD 4 mice, EtOH significantly increased the expression of GRP78, activated ATF6, phosphorylated-IRE1α, phosphorylated-eIF2a, and caspase-12 (Fig. 3b) in the prefrontal cortex [44]. Pre-treatment of ER stress inhibitor 4-PBA 1 day and 0.5 h before subcutaneous alcohol injection significantly alleviated alcohol-induced ER stress and neuroapoptosis in PD 4 mouse brains (Fig. 3b) [44]. These results suggest that ER stress plays an important role in alcohol-induced neurodegeneration in the developing brain and the vulnerability of the immature brain to alcohol neurotoxicity could result from a higher sensitivity to the ER stress.

Fig. 3.

MANF deficiency exacerbates alcohol-induced ER stress and neuronal damage in the developing brain (adapted from previous publications: [44, 45, 60]). a Postnatal day 4 (PD 4) mouse pups received two subcutaneous injections of EtOH (2.5 g/kg or saline) with 2h interval. Eight hours after the first injection, the expression of cleaved-caspase 3 was analyzed by immunohistochemistry. EtOH drastically increased cleaved-caspase 3 in the PD 4 brain in both CC and CB [45]. Scale bar = 500 μm. b Immunoblots of apoptosis and ER stress markers demonstrated that inhibition of ER stress by 4-PBA pretreatment (100 mg/kg) significantly reduced EtOH induced neuronal apoptosis and UPR activation in PD 4 brain. *p < 0.05 [44]. c–d MANF deficiency exacerbated EtOH induced neuronal apoptosis as revealed by immunoblotting (c) and immunohistochemistry (d) of cleaved caspase-3. *p < 0.05 or **p < 0.01 vs. CTL/PBS. #p < 0.05 or ##p < 0.01 vs. CTL/EtOH [60]. Scale bar = 20 μm. e Immunoblots of ER stress markers in the whole brains of PD 7 pups from control and MANF KO groups. *p < 0.05 or **p < 0.01 vs. CTL/PBS. #p < 0.05 vs. CTL/EtOH [60]. f Immunofluorescent staining of CHOP (red) and DAPI (blue) in PD 7 brains from control and MANF KO groups. *p < 0.05 or **p < 0.01 vs. CTL/EtOH [60]. Scale bar = 20 μm. Abbreviations: CC, cerebral cortex; CB, cerebellum; CA1, hippocampus CA1 region; CTL, control; 4-PBA, 4-phenylbutyric acid; Cl. Casp 3, cleaved-caspase 3

Alcohol also induces ER stress in the adult brain. Using the acute alcohol binge model through intragastric gavage in adult mice, we showed that binge alcohol exposure for 5 or more days can induce ER stress in the adult brain, with a concomitant neurodegeneration [47]. We investigated the effects of chronic alcohol consumption on the adult brain using the adult cHAP (crossed high alcohol preferring) mice model. cHAP mice prefer consuming high amounts of alcohol to water when given free access [269]. The blood alcohol concentration (BAC) generated in this model was comparable to that observed in human alcoholics [269]. We demonstrated that long-term (7 months) free access to alcohol led to significant induction of UPR genes expression in the brain, along with neuronal apoptosis, increased oxidative stress, neuroinflammation, decreased thiamine levels, and anxiety-like behavior [270, 271]. Another study showed that 12 weeks of chronic alcohol administration increased the expression of GRP78 and ATF6 in adult mice brains that exhibited reduced Ca²⁺ levels [272].

Other groups have shown that alcohol induces ER stress in aged rodents. Chronic alcohol consumption in aging rats showed declined levels of sarco/endoplasmic reticulum Ca²⁺ (SERCA) pumps, which reduces ER Ca²⁺ concentration and induces smooth ER (SER) dilation in Purkinje cells [273, 274]. Using aged adult rats, Dlugos demonstrated that long-term alcohol exposure (20 and 40 weeks) significantly increased the activity of caspase 12, an ER stress-responsive caspase in the Purkinje cells of cerebellum slices, when challenged with thapsigargin ex vivo [275].

Evidence of MANF’s Protection Against Alcohol-Induced ER Stress and Neuronal Death

As discussed, neuronal MANF is induced in many pathological conditions wherein ER stress is a shared characteristic and MANF is implicated in neuroprotection against neurodegeneration by alleviating ER stress. We therefore hypothesized that MANF is involved in ER stress regulation and neuroprotection against alcohol-induced neurodegeneration. Using a third trimester equivalent mouse model, we have demonstrated that MANF expression was significantly upregulated in the postnatal mouse brain after acute alcohol exposure [43, 44]. Pharmacological inhibition of ER stress was able to reduce alcohol-induced MANF expression in the developing brain [44]. To investigate the role of MANF in alcohol-induced neurodegeneration and its association with ER stress regulation, we recently established a CNS-specific conditional MANF knock-out (cKO) mouse model [60]. A modest increase in ER stress was observed in the brain of cKO mice without notable neurodegeneration [60, 89]. Whole transcriptome RNA sequencing revealed that MANF deficiency altered ER-related RNA signature in the brain with the expression of many protein assessing genes in the ER being altered [60]. Acute alcohol exposure (3 g/kg) resulted in moderate neuroapoptosis in the developing mouse brain of PD 7 mice; MANF deficiency however, significantly exacerbated alcohol neurotoxicity and caused more extensive neurodegeneration (Fig. 3c, d) [60]. To determine whether the increased neuroapoptosis in cKO mice was mediated by ER stress, we examined the expression of UPR genes and found that MANF deficiency promoted the expression of GRP78, ATF6, XBP1s, and CHOP in the cerebral cortex, cerebellum, and hippocampus in response to alcohol (Fig. 3e, f). Taken together, the data suggests that MANF is neuroprotective against alcohol neurotoxicity by functioning as an ER stress-buffering neurotrophic factor and that MANF deficiency aggravates alcohol-induced ER stress and neuronal death. A list of alternative gene names is provided (Table 4).

Table 4.

List of alternative gene names

Gene name	Human gene symbol	Alternate name(s)
Mesencephalic astrocyte derived neurotrophic factor	MANF	ARP; ARMET
Brain derived neurotrophic factor	BDNF	ANON2; BULN2
Glial cell derived neurotrophic factor	GDNF	ATF; ATF1; ATF2; HSCR3; HFB1-GDNF
Cerebral dopamine neurotrophic factor	CDNF	ARMETL1
Ciliary neurotrophic factor	CNTF	HCNTF
Interleukin 6	IL6	CDF; HGF; HSF; BSF2; IL-6; BSF-2; IFNB2; IFN-beta-2
BCL2 apoptosis regulator	BCL2	Bcl-2; PPP1R50
BCL2 like 1	BCL2L1	BCLX; BCL2L; Bcl-X; PPP1R52; BCL-XL/S
BCL2 associated X, apoptosis regulator	BAX	BCL2L4
BCL2 antagonist/killer 1	BAK1	BAK; CDN1; BCL2L7; BAK-LIKE
BCL2 associated agonist of cell death	BAD	BBC2; BCL2L8
BH3 interacting domain death agonist	BID	FP497
BCL2 interacting protein 3	BNIP3	NIP3
Eukaryotic translation initiation factor 2 alpha kinase 3	EIF2AK3	PERK; PEK; WRS
Eukaryotic translation initiation factor 2 subunit alpha	EIF2S1	EIF2; EIF-2; EIF2A; EIF-2A; EIF-2alpha
Inositol-requiring enzyme 1 α	IRE1α	ERN1; IRE1P; IRE1; hIRE1p
Activating transcription factor 6	ATF6	ACHM7; ATF6A
Activating transcription factor 4	ATF4	CREB2; TXREB; CREB-2; TAXREB67
DNA damage inducible transcript 3	DDIT3	CHOP; CEBPZ; CHOP10; CHOP-10; GADD153; AltD-DIT3; C/EBPzeta
Heat shock protein family A (Hsp70) member 4	HSPA4	RY; APG-2; HSPH2; hsp70; hsp70RY; HEL-S-5a; HS24/P52
Heat shock protein family A (Hsp70) member 5	HSPA5	GRP78; BIP; HEL-S-89n
Heat shock protein family A (Hsp70) member 8	HSPA8	LAP1; HSC54; HSC70; HSC71; HSP71; HSP73; LAP-1; NIP71; HEL-33; HSPA10; HEL-S-72p
Heat shock protein 90 beta family member 1	HSP90B1	ECGP; GP96; TRA1; GRP94; HEL35; HEL-S-125m
Ku autoantigen p70 subunit	KU70	XRCC6; ML8; TLAA; CTC75; CTCBF; G22P1
RELA proto-oncogene, NF-kB subunit	RELA	p65; CMCU; NFKB3
Neuroplastin	NPTN	GP55; GP65; SDR1; np55; np65; SDFR1
Tyrosine hydroxylase	TH	TYH; DYT14; DYT5b
Paired box 2	PAX2	FSGS7; PAPRS
Nuclear receptor subfamily 4 group A member 2	NR4A2	NOT; RNR1; HZF-3; NURR1; TINUR
AKT serine/threonine kinase 1	AKT1	AKT; PKB; RAC; PRKBA; PKB-ALPHA; RAC-ALPHA
Mitogen-activated protein kinase 1	MAPK1	ERK; p38; p40; p41; ERK2; ERT1; NS13; ERK-2; MAPK2; PRKM1; PRKM2; P42MAPK; p41mapk; p42-MAPK
Mechanistic target of rapamycin kinase	MTOR	SKS; FRAP; FRAP1; FRAP2; RAFT1; RAPT1
Calreticulin	CALR	RO; CRT; SSA; cC1qR; HEL-S-99n
Homocysteine inducible ER protein with ubiquitin like domain 1	HERPUD1	SUP; HERP; Mif1
X-box binding protein 1	XBP1	XBP2; TREB5; XBP-1; TREB-5
TATA-box binding protein	TBP	HDL4; TBP1; GTF2D; SCA17; TFIID; GTF2D1
Caspase 3	CASP3	CPP32; SCA-1; CPP32B
Caspase 12	CASP12	CASP-12; CASP12P1
Cytochrome P450 family 2 subfamily E member 1	CYP2E1	CPE1; CYP2E; P450-J; P450C2E
Alcohol dehydrogenase 1A (class I), alpha polypeptide	ADH1A	ADH1
Aldehyde dehydrogenase 1 family member A1	ALDH1A1	ALDC; ALDH1; HEL-9; HEL12; PUMB1; ALDH11; RALDH1; ALDH-E1; HEL-S-53e
Aldehyde dehydrogenase 2 family member	ALDH2	ALDM; ALDHI; ALDH-E2

Conclusions and Future Perspectives

MANF is highly expressed in the developing CNS and is required for normal neuronal development, including neuron differentiation, migration, survival, and neurite extension and maintenance. As an ER stress-responsive neurotrophic factor, its expression and secretion are upregulated in response to ER stress. Accumulating evidence in both animal models and cell cultures demonstrated that MANF offers protection in various neurodegenerative disorders, such as PD, AD, cerebral ischemia, and spinocerebellar ataxia by alleviating ER stress. Developmental exposure to alcohol induces ER stress and increases UPR in the developing brain, with a concurrent induction of MANF expression. MANF acts as an ER stress modulator, protecting neurons from alcohol-induced ER stress and cell death. As a result, MANF may have significant therapeutic potential to treat ER stress-related diseases, including FASD. Although several membrane molecules including NPTN, PERK, IRE1α, and ATF6 were shown to be potential MANF transmembrane receptors, the underlying mechanisms of how MANF regulates ER stress and mitigates cell death remain to be elucidated. Further studies are required to dissect the intracellular and extracellular actions of MANF as well as identify MANF interacting molecules and downstream signaling pathways that govern MANF’s function in regulating neuronal development and protecting neurons from ER stress-induced neuronal damage.

Abbreviations

FASD

Fetal alcohol spectrum disorders

FAS

Fetal alcohol syndrome

PAE

Prenatal alcohol exposure

ER

Endoplasmic reticulum

SER

Smooth endoplasmic reticulum

CNS

Central nervous system

MANF

Mesencephalic astrocyte-derived neurotrophic factor

CDNF

Cerebral dopamine neurotrophic factor

NTFs

Neurotrophic factors

BDNF

Brain-derived neurotrophic factor

GFLs

Glial cell line-derived neurotrophic factor family of ligands

GDNF

Glial cell line-derived neurotrophic factor

CNTF

Ciliary neurotrophic factor

IL-6

Interleukin 6

ROS

Reactive oxygen species

UPR

Unfolded protein response

PERK

Pancreatic ER kinase-like ER kinase

IRE1α

Inositol-requiring enzyme 1 α

ATF6

Activating transcription factor 6

XBP1s

X-box binding protein 1

GRP78

Glucose-regulated protein 78 kDa

ERAD

ER-associated protein degradation

PD

Parkinson’s disease

AD

Alzheimer’s disease

ARP

Arginine-rich protein

UTR

Untranslated region

SAPLIP

Saposin-like protein

PDIs

Protein disulfide isomerases

SCG

Superior cervical ganglion

KDELRs

KDEL receptors

TH

Tyrosine hydroxylase

KO

Knock-out

NSCs

Neural stem cells

MCAO

Middle cerebral artery occlusion

ERSE

ER stress response element

AAV

Adeno-associated virus

BMSC

Bone marrow mesenchymal stem cells

6-OHDA

6-Hydroxydopamine

HSP70

Heat shock protein 70

Aβ

Amyloid β-peptide

APP/PS1

Amyloid precursor protein/presenilin 1

ITGC

Inferior temporal gyrus of the cortex

SCA17

Spinocerebellar ataxia 17

TBP

TATA box binding protein

RP

Retinitis pigmentosa

sI/R

Simulated ischemia/reperfusion

MRI

Magnetic resonance imaging

GD

Gestation days

PD

Postnatal days

ADH

Alcohol dehydrogenase

CYP2E1

Cytochrome P450 2E1

ALDH

Aldehyde dehydrogenase

BAC

Blood alcohol concentration

BBB

Blood-brain barrier

4-PBA

4-Phenylbutyric acid

MEFs

Mouse embryonic fibroblasts

Bcl2

Bcl2 apoptosis regulator

Bcl2l1

Bcl2 like 1

Bax

Bcl2 associated X apoptosis regulator

Bak1

Bcl2 antagonist/killer 1

Bad

Bcl2 associated agonist of cell death

Bid

BH3 interacting domain death agonist

Bnip3

Bcl2 interacting protein 3

cHAP

Crossed high alcohol preferring

SERCA

Sarco/endoplasmic reticulum Ca²⁺

ATP

Adenosine tri-phosphate

ADP

Adenosine di-phosphate