Mutant mice with Bip deficiency die during the embryonic period (Luo et al., 2006; Jin et al., 2017), and the main cause of death is abnormal expression of the pulmonary surfactant protein surfactant protein-A (SP-A) and prominently surfactant protein-C (ProSP-C), which subsequently triggers respiratory insufficiency in neonatal or newborn mice and leads to death. In addition to respiratory failure, Bip mutant mice exhibit abnormal neuronal migration and dysplasia. Normal cortical neurogenesis in the ventricular zone (VZ), and newborn neurons migrate to different layers of the cerebral cortex through radial migration (Nadarajah and Parnavelas, 2002; Marín et al., 2010). Neurons generated earlier migrate to the deep cortex, whereas neurons generated later migration to shallow layers (Ghashghaei et al., 2007; Kwan et al., 2012). The subplate (SP), cortical plate (CP), and marginal zone (MZ) are formed in an inside-out migration pattern during development (Caviness, 1982). However, newborn neurons in Bip mutant mice are the first to migrate to the MZ and remain there, whereas later-born neurons fail to migrate to the upper layers. The mutant brain exhibits an outside-in pattern during neocortical layer formation. This phenotype is observed because loss of Bip affects the expression of reelin, a protein that plays an essential role in neuronal migration. In wild-type mice, reelin-secreting Cajal-Retzius (CR) cells are located in the superficial layer of the cortex. In contrast, CR cells in mutant Bip mice are scattered around the upper layer of the neocortical primordium, and CR cells in Bip mutants do not secrete reelin. Therefore, Bip mutant mice exhibit a phenotype similar to that of reelin knockout mice (Mimura et al., 2008).

Heterozygous mutant Bip mice exhibit no significant difference in lifespan compared with wild-type mice, but mutant Bip mice develop renal tubular-interstitial lesions as they age (Kimura et al., 2008). Some mutant Bip mice develop paralysis and tremors after 12 months. These mice exhibit motor nerve injury symptoms, such as loss of righting reflex, and suffer from paralysis (Jin et al., 2014). In addition, studies have shown that Bip deletion is associated with the neurodegenerative disease amyotrophic lateral sclerosis (ALS). ALS is a fatal neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons (Beghi et al., 2011; Filareti et al., 2017). In recent years, impaired protein homeostasis has been found to be a key factor in the pathogenesis of ALS (Zhao et al., 2008; Ruegsegger and Saxena, 2016), and Bip plays an important role in this process. Studies have found that Bip-deficient mice show more severe neurological decline and spinal motor neuron loss symptoms than normal mice. Gómez-Almería et al. created mutant superoxide dismutase 1 (mSOD1) and Bip double mutant (mSOD1/Bip+/-) mice to study the role of Bip in a mouse model of the neurodegenerative disease ALS (Gómez-Almería et al., 2021). They found that knockout of Bip show more intense neurological decline than mSOD1 single knockout mice. Then, to determine the relationship between the Bip protein and the pathogenesis of ALS, they also discussed the potential relationship between the Bip protein and the type-1 cannabinoid (CB1) receptor that mediates neuroprotection. CB1 receptor is mainly located in neurons of the central nervous system (Chiarlone et al., 2014). Numerous studies have reported that CB1 receptor-mediated neuroprotection is related to ALS (Abood et al., 2001; Rossi et al., 2010). They found that in mSOD1 mice with partial Bip gene deletion, the level of the CB1 receptor was significantly decreased, which indirectly indicated that Bip may play a neuroprotective role in ALS (Gómez-Almería et al., 2021). Similarly, Apolloni et al. found that increasing Bip and Hsp70 protein levels in the spinal cord and cortex of ALS model mice could rescue the loss of motor neuron dendritic spines (Apolloni et al., 2019). These studies suggest that Bip plays vital roles in motor neuron development.

Studies have shown that the interaction between Bip and N-methyl-D-aspartate (NMDA) receptors may affect synaptic transmission. NMDA receptors play essential roles in brain development, plasticity, and pathology. Functional NMDA receptors are tetramers mainly composed of two GluN1 subunits and two GluN2 regulatory subunits (Cull-Candy et al., 2001; Yashiro and Philpot, 2008). The number and composition of GluN2A and GluN2B containing NMDA receptors at synapses are dynamically regulated during development and neuronal activation, which is thought to control synaptic plasticity (Nase et al., 1999; Lopez de Armentia and Sah, 2003; Liu et al., 2004). Using immunoprecipitation, the authors examined the distribution of Bip in neurons and found that Bip is strongly expressed in both cortical and hippocampal neuronal cell bodies and neurites. Furthermore, Bip co-localizes with GluN2A in cultured cortical neuron dendrites, and preventing the binding of Bip to GluN2A reduces synaptic transmission and impairs memory formation (Zhang et al., 2015).

SIL1 is a 54-kDa ER protein composed of 461 amino acids and is a NEF for Bip (Wang J. et al., 2017). As an auxiliary protein of Bip, the synergistic effect of SIL1 and Bip has consistently been a research hotspot. The central nervous system of mice with SIL1 deficiency exhibits developmental defects, including abnormal neuronal morphological development, cortical neuron migration, and localization (Inaguma et al., 2014).

Denzel et al. found that approximately 50% of CNX knockout mice died within 48 h after birth, most of the remaining mice died within 4 weeks, and only a few mice survived to 3 months (Denzel et al., 2002). CNX gene knockout (cnx^–/–) mice exhibited significant movement impairment, including unsteady gait, inactive walking, and obvious trunk ataxia. The authors then examined the sciatic nerve in the gene-deficient mice through electron microscopy and found that most of the mice exhibited a reduced number of medullary nerve fibers and a reduced sciatic nerve diameter (Denzel et al., 2002). However, the cause of the short-lived death of cnx^–/– mice has not yet been discovered but might be related to the coordinating role of CNX proteins in development. This hypothesis deserves further exploration.

Recent studies assessing the localization of CNX and NMDA receptors on neurons based on specific immunostaining and biotin chemical reagent labeling revealed that a considerable portion of CNX was localized on the cell surface of cultured neurons, and this localization was regulated by NMDA receptors (Itakura et al., 2013). These results suggest that CNX may play an essential role in NMDA receptor-mediated neuronal function. Other studies demonstrated that α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors interact with CNX and Bip in neuronal cell bodies and hippocampal pyramidal neuron dendrites (Rubio and Wenthold, 1999; Gerrow and Triller, 2010). In addition, the AMPA receptor is a tetramer composed of four subunits, GluA1, GluA2, GluA3, and GluA4. This receptor is expressed in both neurons and glial cells (Pick and Ziff, 2018). It is well known that NMDA receptors and AMPA receptors play essential roles in central nervous system development. NMDA receptors are involved in experience-dependent synapse and dendritic development (Nadarajah and Parnavelas, 2002; Luo et al., 2006), whereas AMPA receptor expression directly affects neuronal dendritic complexity, synaptic maturation, and neural network formation (Buffington et al., 2014). Defects in AMPA receptor expression are the underlying causes of developmental diseases, degenerative diseases, and cognitive dysfunction (Kessels and Malinow, 2009; Henley and Wilkinson, 2016; Guo and Ma, 2021). However, the specific effects of CNX loss on NMDA receptor or AMPA receptor membrane expression or function remain to be investigated. Moreover, studies have shown that CNX can be attached to membranes through NMDA receptor-mediated pathways (Itakura et al., 2013). Therefore, CNX may regulate central development by participating in receptor function at the cell membrane. In addition, CNX regulates calcium levels in the ER (Roderick et al., 2000), which subsequently regulates receptor transport to influence synaptic strength (Turrigiano, 2008). Therefore, CNX may directly affect synaptic strength and synaptic development through this mechanism. However, the direct correlation between CNX and synaptic development and function needs to be expanded.

Activity-regulated cytoskeleton-associated (Arc) protein is a master regulator of long-term synaptic plasticity and memory formation (Bramham et al., 2010; Korb and Finkbeiner, 2011; Shepherd and Bear, 2011; Nikolaienko et al., 2017). Arc proteins control synaptic strength by promoting AMPA receptor endocytosis (DaSilva et al., 2016) and activity-dependent long-term changes in synaptic strength (Ehlers, 2000; Newpher and Ehlers, 2008). Arc overexpression decreased the density of GluA1 subunits of AMPA receptors on the cell surface and AMPA receptor-mediated miniature excitatory post-synaptic currents (mEPSCs). Arc mutations that prevent clathrin-adaptor protein 2 (AP-2) interaction reduce Arc-mediated endocytosis of GluA1 and abrogate AMPA receptor-mediated mEPSCs reduction (DaSilva et al., 2016). Endocytosis is the process by which cells internalize substances through the plasma membrane. Endocytosis plays an important role in different stages of the development of the nervous system, such as the formation of continuous synaptic transmission (Sudhof, 2004), the entry of drugs into the central nervous system (Smith et al., 2008), and the internalization of receptor-ligand complexes to nerve endings for intracellular signaling (Gloor et al., 2001). CNX is also involved in clathrin-mediated endocytosis of neurons (Li et al., 2011). Kraus et al. performed an electron microscopy analysis of the cerebellum of 7-day-old wild-type and CNX-null mice to assess whether CNX is involved in neuronal endocytosis. The results showed that the number of synaptic vesicles was significantly increased in the cerebellum of CNX-deficient mice compared with wild-type mice, demonstrating that CNX deficiency leads to increased endocytic activity in the mouse neuronal system (Kraus et al., 2010). Recent studies have found a direct interaction between Arc and CNX in neurons, and the interaction between recombinantly expressed glutathione S-transferase (GST)-tagged Arc and endogenous CNX has also been found in HEK293, SH-SY5Y neuroblastoma, and PC12 cells (Myrum et al., 2017). Given that both Arc and CNX mediate clathrin-dependent endocytosis and that both are expressed in excitatory synapses, it is possible that Arc and CNX cooperate in regulating endocytosis. Therefore, whether CNX is involved in the synaptic effect is also a mechanism that needs further exploration.

To study the role of CRT during development in vivo, Rauch et al. generated CRT-deficient mice by targeted inactivation of the CRT gene. The results demonstrated that CRT heterozygous mice were viable and fertile, whereas crt^–/– mice were embryonic lethal and exhibited multiple disease phenotypes, including cardiomyopathy, anencephaly, and omphalocele (Rauch et al., 2000). In addition, CRT is also important for cranial neural tube closure and is highly expressed in the developing brain during neurogenesis (Mesaeli et al., 1999). Neural tube formation is a complex process that requires the interaction of neuroepithelial cells and the underlying mesenchyme (Lynch et al., 2012; Correll et al., 2019). Disruption of neural tube closure was found in several CRT knockout mice, in which proteins involved in neuronal migration and calcium signaling were inactivated (Solheim et al., 1997; Rauch et al., 2000). These results demonstrate that CRT is important for embryonic brain development, especially the neural tube development. However, it is currently unclear which proteins interact with CRT to regulate embryonic neural development.

Neurotrophic factors, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor-3 (NT-3), and neurotrophic factor-4/5 (NT-4/5), are key tissue factors that control nervous system development (Chao, 2003). The NGF/tyrosine receptor kinase A (TrkA) signaling pathway is necessary for neural development, and abnormalities in this pathway can lead to abnormal neuronal differentiation. Shih et al. found that in NGF-stimulated PC12 cell differentiation, CRT levels were increased by activation of mitogen-activated protein kinase (MAPK) mediated by extracellular signal-regulated kinase (ERK). CRT deficiency significantly reduced NGF-induced neuronal differentiation. Furthermore, CRT overexpression enhanced neuronal differentiation through simultaneous activation of the ERK-dependent MAPK pathway (Shih et al., 2012). The Ca²⁺ regulation ability of CRT is also essential for NGF-triggered neuronal differentiation (Pilquil et al., 2020). In addition, studies have shown that CRT is a novel oncogenic N-MYC (MYCN) inhibitor that can down-regulate MYCN promoter activity and protein expression to regulate neuronal differentiation. This study also found that CRT-mediated MYCN inhibition resulted in increased neurite length (Lee et al., 2019).

Calreticulin localizes to axons of cultured dorsal root ganglion neurons in vitro and peripheral nervous system axons in vivo (Willis et al., 2005; Vuppalanchi et al., 2010), and automatic axonal regeneration occurs after injury to adult peripheral neurons (Bradke et al., 2012). The intrinsic growth capacity of peripheral nervous system axons after injury is greater than that of central nervous system axons. Pacheco et al. constructed in vitro and in vivo neuronal axonal injury models to study the relationship between CRT and axonal regeneration after axonal injury. Intra-axonal CRT levels were elevated after peripheral nerve injury in vivo. Unilateral sciatic nerve crush injury was performed in the mid-thigh of male SD rats, and then sciatic nerve sections were collected and stained 1 h, 6 h, 18 h, and 7 days after the crush injury. Compared with the control sciatic nerve, axonal CRT levels were significantly increased 6 h after injury and reached a maximum at 18 h after injury. The authors then performed axotomy on dorsal root ganglion neurons to study the effect of CRT deletion on the post-operative axonal growth process. The results showed that the shRNA-mediated CRT deletion group exhibited exacerbated post-axotomy contractions. In contrast, axon-targeted expression of unrestricted CRT mRNA reduces contraction and promotes axon regeneration after axotomy in vitro (Pacheco et al., 2020). In conclusion, the overexpression of axon-targeted CRT promotes axon regeneration after axonal injury, indicating that CRT plays an important role in axonal growth.

Protein disulfide isomerase-1 controls neuronal migration by regulating Wnt secretion. PDI is a family of protein chaperones that reside in the ER and are capable of catalyzing the formation (oxidation), cleavage (reduction), and rearrangement (isomerization) of disulfide bonds (Wilkinson and Gilbert, 2004). The formation of a proper Wnt protein structure requires the formation of disulfide bonds, which are critical for Wnt secretion and signaling (Zhang X. et al., 2012; MacDonald et al., 2014). Wnt proteins play essential roles in neuronal migration and axon-dendritic guidance in vertebrates (Yoshikawa et al., 2003; Bocchi et al., 2017), and they also control neural developmental processes in C. elegans, including neuronal migration, polarity, and axon guidance (Coudreuse et al., 2006; Hilliard and Bargmann, 2006; Pan et al., 2006). Therefore, abnormal Wnt expression or function significantly impairs neural development.

PDI-1 controls the secretion of EGL-20, the Wnt homolog in C. elegans, and therefore affects neuron migration (Torpe et al., 2019). EGL-20/Wnt is expressed and secreted in subcutaneous (epidermal) and muscle cell subsets of C. elegans (Whangbo and Kenyon, 1999). EGL-20 function is critical for the migration of hermaphroditism-specific neuron (HSN) (Desai et al., 1988). Torpe et al. used HSN development as a readout of EGL-20 function to identify molecules that control Wnt maturation and secretion. They hypothesized that PDI is important for EGL-20-directed HSN development. The expression of five PDI-encoding genes (pdi-1, pdi-2, pdi-3, pdi-6, and C14B9.2) in C. elegans was reduced using RNA interference, and the development of HSN was analyzed. This study found a specific requirement for PDI-1 during HSN development with approximately 20% of PDI-1-silenced animals exhibiting a phenotype deficient in HSN migration. Subsequently, to determine whether PDI-1 also controls EGL-20-dependent post-embryonic neuron development, the authors performed post-embryonic neuronal migration assays in PDI-1-deficient animals and found that PDI-1 knockout animals exhibited migration defects (Torpe et al., 2019). PDI inhibitors reduce Wnt3a secretion in human cells. Most mammalian genomes contain 19 Wnts and greater than 20 PDI proteins, which exhibit distinct expression domains and biological functions (Ellgaard and Ruddock, 2005; Galligan and Petersen, 2012; Willert and Nusse, 2012). Mammalian Wnt3a secretion was abolished when a single cysteine residue was replaced by alanine (MacDonald et al., 2014), suggesting that Wnt secretion is influenced by disulfide coordination. These results suggest that PDI can affect neuronal migration by regulating the secretion of Wnt and its homolog EGL-20.

The role of the Wnt signaling pathway in early cortical development is not limited to the migratory localization of neurons. It is also involved in neurogenesis, neuronal differentiation, and axon-dendritic guidance (Freese et al., 2010; Munji et al., 2011). In addition, the atypical Wnt signaling pathway is involved in the assembly of neural circuits (Hinata et al., 2007; Wang et al., 2011), especially dendrite development and synaptogenesis in cultured pyramidal neurons (Salinas and Zou, 2008). Wnt pathway dys-regulation is closely related to human neurological diseases (Castelo-Branco et al., 2003). However, the mechanism by which PDI further affects other developmental processes by affecting the Wnt signaling pathway is unclear and deserves further exploration.

Sigma-1 receptor regulates neurite outgrowth (Ishima et al., 2014) as well as primary neuron morphogenesis (Ishima and Hashimoto, 2012). Dendritic spines in hippocampal neurons play important roles in neuroplasticity and memory formation. siRNAs were used to silence the expression of Sig-1R to study their role in the morphogenesis of primary hippocampal neurons in vitro. The results showed impaired dendrite extension and branching after Sig-1R gene silencing. Moreover, Sig-1Rs also have a major impact on the formation and maturation of dendritic spines in later stages (Tsai et al., 2009). In rat hippocampal primary neurons, reducing the expression of Sig-1Rs by siRNA resulted in defects in dendritic spine formation. Tsai et al. observed and analyzed the dendritic spine morphology and maturity of cultured hippocampal neurons. On DIV 16, the control group neurons formed filamentous protrusions on the dendrites. On DIV 22, control neurons formed clusters of short, thick mushroom-like spine heads without filopodia, indicating spine maturation. In contrast, Sig-1R-siRNA-transfected (siSig-1R-tf) neurons formed elongated protrusions with a few spine heads at the tips. The longer the culture time, the more pronounced the abnormal phenotype. This finding suggests that Sig-1Rs are endogenous regulators of hippocampal dendritic spine formation (Tsai et al., 2009).

Sigma-1 receptor also plays an important role in the development of synaptic function (Ryskamp et al., 2019). Active synapses express specific proteins and receptors for proper function. Studies have shown that Sig-1R knockdown inhibits functional synapse formation. GluA2, GluA3, and PSD-95 staining revealed a strong immune response on dendritic spines in control neurons. However, this response was not observed in filopodia protrusions of siSig-1R-tf neurons, whereas these proteins were still present in dendritic shafts. The axonal terminals of siSig-1R-tf neurons also showed significantly reduced immune responses to synaptophysin. Neurons labeled with FM4-64, a membrane-selective red fluorescent dye, were depolarized with KCl to measure synaptic activity. The results showed that siSig-1R-tf neurons did not form functional synapses. These results suggest that Sig-1Rs have essential effects on synapse formation and synaptic function (Ryskamp et al., 2019).

Earlier, we mentioned that the NMDA receptor is associated with learning and memory. A study found that changes in Sig-1R can cause changes in NMDA receptor expression. Sig-1R was overexpressed by intraperitoneal injection of the Sig-1R activation agonist SKF10,047 in SD rats, and subsequent immunofluorescence staining revealed increased GluN2A, GluN2B, and PSD95 expression (de Montigny et al., 1992; Monnet et al., 1994, 1996). This effect was abolished after the addition of the Sig-1R antagonist BD1063 (Matsumoto et al., 1995; McCracken et al., 1999). In addition, they found increased interactions between Sig-1R and GluN2 subunits as well as increased surface levels of NMDA receptor upon Sig-1R activation, which suggests that Sig-1R regulates the expression of NMDA receptor and related neural function (Pabba et al., 2014).

The membrane of the ER of a cell forms contacts directly with mitochondria, and the contact is referred to as the mitochondrion-associated ER membrane (MAM) (Mori et al., 2013). The MAM is a subdomain of the ER that engages in a direct physical association with mitochondria (Rizzuto et al., 2004; Csordás et al., 2006; Szabadkai et al., 2006). The MAM integrates many signaling pathways and is important for cellular survival because it serves as the “tunnel” for lipid transport and Ca²⁺ signaling between the ER and mitochondria (Cárdenas et al., 2010). Sig-1Rs are ER chaperones that localize specifically at the MAM and regulate a variety of cellular functions including Ca²⁺ signaling between ER and mitochondria, neuronal differentiation, ion channel activities, and notably cellular survival (Aydar et al., 2002; Fontanilla et al., 2009; Fukunaga and Moriguchi, 2017). However, exactly how Sig-1R regulates the neuronal differentiation process at the MAM is not fully clarified. Neurons are highly differentiated cells and require great amounts of ATP for the maintenance of cell membrane ionic gradients and neurotransmission (Xavier et al., 2016). Mitochondria, the main intracellular energy transducers, play a key role in neural development by producing ATP and biosynthetic substrates, regulating Ca²⁺ homeostasis, and initiating apoptosis (Kann and Kovács, 2007). Given the vital role of Sig-1R in the MAM and the importance of energy transport for neural development, exploring the energy transduction mechanism of Sig-1R in the MAM may be key to studying the developmental process.

Autism spectrum disorder is a brain developmental disorder characterized by language disorders, social difficulties, and repetitive and stereotyped behaviors (Lai et al., 2014).

Intellectual disability (ID) is a common disorder of nervous system development that leads to intellectual disabilities. Various factors can cause ID, including loss of important synaptic function, abnormal neuronal morphological development, and neuronal migration and localization disorders (Kaufman et al., 2010; Ropers, 2010; Mehregan et al., 2016).

Bipolar disorder is a common mental disorder characterized by recurrent episodes of mania, hypomania, and depression. BD is characterized by mood swings between elevated and depressed moods (Rosso et al., 2007). The exact mechanism of these mood swings remains unclear and needs to be further elucidated.

Attention-deficit hyperactivity disorder is a syndrome characterized by inattention, hyperactivity, emotional impulsivity, and learning difficulties (Tripp and Wickens, 2009).

At present, there are few studies on the role of molecular chaperones in ADHD, and more relevant studies exist at the level of drug treatment targets. Methylphenidate (MPH) is a commonly used stimulant drug for ADHD (Capp et al., 2005; Arnsten, 2006). The symptoms of ADHD patients are mostly consistent with dysfunction in the pre-frontal cortex (PFC) (Barkley et al., 1992), a highly functional region that directs and organizes attention, thoughts, and emotions (Arnsten and Li, 2005). It has been demonstrated that low-dose MPH injection into the PFC can improve working memory performance (Arnsten and Dudley, 2005). High MPH doses can cause behavioral sensitization and a high risk of addiction (Amini et al., 2004). Sig-1R receptor proteins are highly distributed in the PFC, striatum and hippocampus. At the cellular level, Sig-1R is post-synaptic (Hayashi and Su, 2004) and is an excitatory target for the treatment of ADHD (e.g., cocaine) (Matsumoto et al., 2001; Maurice et al., 2002). MPH was found to increase NMDA receptor-mediated synaptic transmission, which was mediated by Sig-1R. Under the same dose of MPH stimulation, the NMDA-induced post-synaptic strength in the hippocampus of mice supplemented with the Sig-1R agonist PRE-084 was significantly higher than that of the control group (Motawe et al., 2020). Similarly, pretreatment with the Sig-1R antagonist BD1063 effectively prevented hyperactivity caused by MPH hyperactivity (Matsumoto et al., 1995), confirming the importance of Sig-1R receptors in excitatory synaptic transmission (Zhang C. L. et al., 2012). Therefore, the active use of Sig-1R receptor ligands in the treatment of ADHD and prevention of MPH-induced addiction may have unexpected effects.