Role of Seipin in Human Diseases and Experimental Animal Models

Seipin, a protein encoded by the Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) gene, is famous for its key role in the biogenesis of lipid droplets and type 2 congenital generalised lipodystrophy (CGL2). BSCL2 gene mutations result in genetic diseases including CGL2, progressive encephalopathy with or without lipodystrophy (also called Celia’s encephalopathy), and BSCL2-associated motor neuron diseases. Abnormal expression of seipin has also been found in hepatic steatosis, neurodegenerative diseases, glioblastoma stroke, cardiac hypertrophy, and other diseases. In the current study, we comprehensively summarise phenotypes, underlying mechanisms, and treatment of human diseases caused by BSCL2 gene mutations, paralleled by animal studies including systemic or specific Bscl2 gene knockout, or Bscl2 gene overexpression. In various animal models representing diseases that are not related to Bscl2 mutations, differential expression patterns and functional roles of seipin are also described. Furthermore, we highlight the potential therapeutic approaches by targeting seipin or its upstream and downstream signalling pathways. Taken together, restoring adipose tissue function and targeting seipin-related pathways are effective strategies for CGL2 treatment. Meanwhile, seipin-related pathways are also considered to have potential therapeutic value in diseases that are not caused by BSCL2 gene mutations.


Introduction
Seipin, an evolutionarily conserved protein encoded by the Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) gene, is found to locate in the endoplasmic reticulum (ER) [1,2]. SEIPIN was initially discovered in type 2 congenital generalised lipodystrophy 2 (CGL2) patients, implying that it plays an important role in adipose tissue homeostasis [2]. Lipid droplets (LDs), the main organelles for storing triacylglycerols (TAGs) in adipose tissue, play a key role in lipid homeostasis and normal cell physiology [3]. Seipin belongs to an array of ER proteins including fat-storage-inducing transmembrane protein 2 (FIT2), Atlastin, receptor-expression-enhancing protein 1 (REEP1), etc., that are involved in LD biogenesis. Seipin deficiency can result in abnormalities of LDs in terms of number and morphology, which eventually lead to the loss of adipocyte tissue [4]. Therefore, the structure and function of seipin have been increasingly researched and summarised [5][6][7].

Structure and Physiological Function of Seipin
The N-terminal and C-terminal of the ER-located seipin protein are exposed to the cytoplasm, and the two transmembrane helices wrapped in the ER lumen are connected by a large, highly conserved cavity ring [22]. Multiple seipin subunits form a cyclic protein complex [23], and the hydrophobic α-helices at the inner rim of the seipin protein complex are likely binding to the ER membrane [23,24]. While they remain elusive, existing studies have outlined the predominant functions of seipin as follows: (1) Seipin interacts with proteins involved in TAG synthesis to ensure normal adipogenesis [25,26]. Firstly, seipin deficiency significantly elevates glycerol-3-phosphate acyltransferase (GPAT) activity, resulting in retarded adipogenesis [25]. Secondly, seipin could bind to 1-acylglycerol-3phosphate O-acyltransferase 2 (AGPAT2), phosphatidic acid (PA) phosphatase lipin 1, or both to regulate the PA metabolism [26]. The binding of seipin simultaneously to AGPAT2 and lipin 1 may facilitate PA clearance by supplying PA from AGPAT2 to lipin 1 [26]. Therefore, seipin deficiency may lead to abnormal PA accumulation [27][28][29]. On the nuclear envelope, the accumulation of PA traps and inhibits peroxisome proliferator activated receptor gamma (PPARγ) and thus blocks adipogenesis [30,31]. The lack of binding between seipin and AGPAT2 also contributes to reduced nuclear PPARγ accumulation, but the underlying mechanism requires further investigation [26]. On the ER, the accumulation of PA increases surface tension and decreases line tension of local ER, impeding unidirectional LD budding and normal ER-LD contact [32]. (2) The structural role of seipin in TAG and diacylglycerol (DAG) facilitates TAG phase separation, LD budding, and growth [33][34][35]. Seipin protein complex not only captures TAG and DAG inside its ring, but also provides TAG clusters for multiple nucleation sites [33,34]. Recently, the seipin protein complex has also been found to form a flexible cage-like structure, and the dynamic conformation changes in this structure contribute to the TAG phase separation, as well as LD growth and budding [35]. (3) Compelling evidence indicates that seipin plays a key role in the structure of ER-LD contact to promote the biogenesis or maintenance of LDs [4,[36][37][38][39]. Seipin facilitates LD initiation via connecting nascent LDs to ER and stabilising ER-LD contact to restrict the lateral movement of LDs [4,38]. Then, seipin promotes LD growth by transferring the cargo of ER proteins and lipids into nascent LDs [38]. Thereafter, seipin further prevents the shrinkage of LDs during TAG flow from smaller to larger LDs, a process called ripening [35], through mobilising TAGs to small LDs [4,37]. Moreover, Fld1 (yeast seipin) cooperates with its partner Pex30 to promote LDs budding and pre-peroxisomal vesicle budding by stabilising ER domains [39]; the Fld1/Ldb16 complex establishes a diffusion barrier, which is necessary for LD surface tension and identity via stabilising ER-LD contacts to avoid phospholipid packing defects and abnormal LDs formation [40]. (4) Seipin promotes fat storage in adipose tissue by regulating calcium homeostasis and mitochondrial energy [41][42][43]. Seipin is enriched in ER-mitochondria contact sites nearby calcium regulators (sarco/ER calcium ATPase pump 2, inositol 3 phosphate receptor, and voltage-dependent anion channel), and interacts with them in a nutritionally regulated manner [43]. Therefore, seipin deficiency results in mitochondrial calcium import defects, leading to impairment of the Krebs cycle [42,43]. Since citrate and ATP are critical for lipogenesis and adipocyte properties, respectively, defects in citrate and ATP suppress fat storage [42,43]. (5) Seipin deficiency also compromises adipocyte properties via inducing ER stress in adipocyte tissue [43,44]. (6) Seipin deficiency accelerates lipolysis. This phenomenon is only observed during the early stage of seipin loss because there are rare TAGs left after this period [43,45,46]. Elevated adipose triglyceride lipase (ATGL) stability and expression, increased phosphorylation of hormone-sensitive lipase (HSL) and perilipin 1 (PLIN1) contribute to seipin deficiency-induced lipolysis acceleration [47,48]. Additionally, seipin has been found to participate in maintaining metabolic homeostasis via recruiting remodelling actin cytoskeleton of adipocytes [49], protecting mature adipocyte tissue [44,46], and restricting lipogenesis as well as LD accumulation in non-adipocyte tissue [50].
Physiologically, seipin has tissue-specific functions. In the adipose tissue, seipin serves as an adipocyte safeguard that maintains normal neutral lipid storage. In the liver, seipin is crucial for hepatic intracellular TAG accumulation [16]. Interestingly, despite adipose tissue and the liver storing neutral lipid in physiological conditions, seipin adipose-specific knockout (seipin-aKO) and liver-specific knockout (seipin-lKO) mice show distinctively different phenotypes. Seipin-aKO mice show overt lipodystrophy and hepatic steatosis [44], while seipin-lKO mice neither develop hepatic steatosis even under a high-fat diet, nor have worsened metabolic disorders on top of a seipin-aKO background [51,52]. Findings from seipin-aKO and seipin-lKO mice suggest that the normal TAG storage function of adipose tissue protects against liver steatosis. In the central nervous system, seipin is also widely expressed and plays a key role in intellectual development and motor neuron function [53]. It has been shown that seipin contributes to the deranged metabolism of phospholipids in the brain: seipin deficiency induces altered phospholipid components in the membrane, which may be associated with defective synaptic vesicle budding; seipin deficiency may also impact synaptic vesicle formation via affecting phospholipids, a main component of synaptic vesicles [54]. Furthermore, seipin has been linked to pre-peroxisomal vesicle biogenesis, which protects neurons from abnormal neuronal migration and differentiation, demyelination, inflammation, oxidative stress, and so on [55,56]. In the testis, seipin is essential for normal spermiogenesis by maintaining testicular phospholipid metabolic homeostasis [14].

Human Diseases Caused by Mutations of BSCL2
SEIPIN is widely expressed in human tissues, and the highest levels of this protein are found in the adipose tissue, brain, and testis [2,57]. Strikingly, the expression of SEIPIN in the brain is inversely correlated with age, but strongly and positively associated with anti-oxidative stress enzymes such as (superoxide dismutase 1 and 2 and PPARγ) [57]. Therefore, the mutations of BSCL2 usually induce a variety of serious clinical consequences ( Figure 1, Table 1 and Supplementary Table S1), including CGL2 (OMIM #269700; highest prevalence, estimated to be 0.1-5 persons per million [45,58]), PELD (OMIM #615924; only nine patients reported worldwide so far) [59], and BSCL2-associated motor neuron diseases (OMIM #619112 or OMIM #270685; secondary prevalence).

CGL2
CGL is a metabolic disease characterised by almost complete loss of adipose tissue and severe metabolic disorders, including early-onset diabetes mellitus, insulin resistance, hypertriglyceridaemia, and hepatic steatosis [58]. Variants of four different genes (AGPAT2, BSCL2, CAV1, and PTRF) induce CGL1, CGL2, CGL3, and CGL4, respectively. All subtypes of CGL lose metabolically active adipose tissue, but each of the subtypes has their unique features in addition to genetic testing (gold criteria) for differential diagnosis: (1) CGL1 patients usually suffer from focal lytic lesions in long bones after puberty; (2) CGL2 patients lose their mechanical adipose tissue except for metabolically active adipose tissue; (3) CGL3 patients are characterised by short stature, functional megaoesophagus, and hypocalcaemia; (4) CGL4 patients may progressively lose their adipose tissue during infancy, and face the challenge of congenital myopathy with high serum levels of creatine kinase [58]. CGL2 accounts for 50.3% of all four CGL distinct subtypes and has the most serious clinical consequence [58,72]. Up to now, there have been no reports of phenotypic differences among different mutation forms in CGL2 patients [58], but a few studies suggest that the severity of CGL2 is associated with sex, with females being more severely affected [73,74]. Since CGL2 patients have systemic multi-organ damage, we endeavour to characterise the phenotype of CGL2 patients from metabolic disorders and their complications, physical features, neurological symptoms, reproductive system symptoms, and others ( Table 2). mutations produce mutant seipin, and mutant seipin induces CGL2, PELD, and BSCL2-associated motor neuron diseases. CGL2 is characterised by metabolic disorders and its complications, which may be caused by adipose tissue loss. In addition, phenotypes resulting from seipin deficiency in breast, testicles, and neurons, including reduced milk production, teratozoospermia, and intellectual impairment, are also observed in CGL2. PELD is a fatal paediatric neurodegenerative disease. BSCL2-associated motor neuron diseases are predominantly damaging motor neurons, resulting in weakness and atrophy of distal muscle groups (hands and legs), foot deformity (pes cavus), and pyramidal tract signs. Abbreviations: BSCL2: Berardinelli-Seip congenital lipodystrophy type 2; CGL2: type 2 congenital generalised lipodystrophy; PELD: progressive encephalopathy with or without lipodystrophy; PCOS: polycystic ovary syndrome; IUGR: intrauterine growth restriction.

Figure 1. Clinical features and organs damaged in diseases caused by BSCL2 mutations. BSCL2
mutations produce mutant seipin, and mutant seipin induces CGL2, PELD, and BSCL2-associated motor neuron diseases. CGL2 is characterised by metabolic disorders and its complications, which may be caused by adipose tissue loss. In addition, phenotypes resulting from seipin deficiency in breast, testicles, and neurons, including reduced milk production, teratozoospermia, and intellectual impairment, are also observed in CGL2. PELD is a fatal paediatric neurodegenerative disease. BSCL2-associated motor neuron diseases are predominantly damaging motor neurons, resulting in weakness and atrophy of distal muscle groups (hands and legs), foot deformity (pes cavus), and pyramidal tract signs. Abbreviations: BSCL2: Berardinelli-Seip congenital lipodystrophy type 2; CGL2: type 2 congenital generalised lipodystrophy; PELD: progressive encephalopathy with or without lipodystrophy; PCOS: polycystic ovary syndrome; IUGR: intrauterine growth restriction.

Changes in teeth and bone
Severe crowding of maxillary and mandibular arches, labial exclusion of 33 and 43, aberrant crown morphology, caries, several teeth missing, generalised plaque induced gingivitis, bone cysts, advanced bone age in the first year of life, hyper-density of partial bones, skeletal abnormalities, diffuse osteosclerosis, well-defined osteolytic lesions sparing the axial skeleton, high serum sclerostin, good bone microarchitecture, high bone mineral density in trabecular sites, multiple bone lytic and pseudo-osteopoikilosis lesions limited to the hands and feet

Others
Developmental delay, multinodular goitre, failure to thrive, significant developmental delay, accelerated growth, chronic nasal congestion, enlarged tonsils and adenoids, voracious appetite, gum bleeding repeatedly, advanced bone age, pancreatitis, upper respiratory tract infection, elevated urinary organic acid, growth disorder, constipation, thrombocytopenia, cardiovascular autonomic neuropathy, papillary thyroid carcinoma Compared with other types of CGL, the loss of adipose tissue in patients with CGL2 is more serious [75,76]. In addition to the absence of metabolically active adipose tissue, a paucity of mechanical adipose tissues in palms, soles, eyes, scalp, and areas around joints is also noted in CGL2 patients [75]. Therefore, CGL2 patients suffer from earlier onset of diabetes mellitus, more severe insulin resistance, and a higher risk of premature death than other forms of CGL [61,62,77]. Recently, a study revealed that lipid peroxidation is increased in blood from CGL2 patients, which may be caused by an imbalance of redox homeostasis and increased mitochondrial DNA damage levels [78]. Cardiac complications and renal injury induced by metabolic disturbance are usually observed in CGL2 patients [58,79]. Cardiac complications in CGL2 patients are prominently characterised by concentric left ventricular hypertrophy, which is associated with diastolic dysfunction but preserved systolic function and ultimately leads to ventricular dysfunction and heart failure [48,80]. Additionally, cardiac complications in CGL2 patients have higher morbidity (42.9%) and poorer prognosis than other types of CGL, which is more likely to cause heart failure and death [58,61,[81][82][83]. CGL is usually associated with proteinuria and renal injury [84]. Of 11 patients with CGL2, 4 developed nephropathy in a study from Turkey [63]. In another study, 2 out of 45 CGL2 patients died from renal failure [60]. Renal anomaly (absence of one kidney) and bilateral renal hypertrophy have also been reported [85,86], but these may be due to individual differences because only one case of each problem has been reported. In addition to hepatomegaly caused by hepatic steatosis, splenomegaly is also usually observed in CGL2 patients [85,[87][88][89][90]. Further, acute pancreatitis has also been reported in a 4-month-old CGL2 infant who has concomitant extremely high serum triglyceride levels (31.9 mmol/L) [82]. High bone mineral density has also been noted in CGL2 patients, and loss of marrow adipose tissue, severe insulin resistance, and low adipokines levels may account for this phenomenon [64,91,92].
Apart from the typical features of metabolic disturbances, CGL2 patients also have a greatly increased probability of developing mild to moderate mental disability [58,60,61,79,95]. Occasionally, symptoms of motor neuron impairment such as waddling gait [60], spastic gait [95], and developmental language disorders [93] have been reported.

PELD
PELD is a fatal paediatric neurodegenerative disease manifested as severe progressive neurodegeneration and concomitant premature death caused by status epilepticus or pneumonia [59]. PELD is induced by a special nonsense mutation-c.985C > T in the BSCL2 gene, and the mutation can be homozygous or in compound heterozygosity with c.507_511del, c.538G > T, or c.1004A > C [1,103]. Recently, Sánchez-Iglesias et al., have comprehensively summarised the diagnosis, treatment, and mechanism of PELD [59]. The mutation can be homozygous or in compound heterozygosity with other mutations

BSCL2-Associated Motor Neuron Diseases
Single heterozygous, compound heterozygous, or homozygous missense mutation (N88S or S90L) in BSCL2 causes a broad spectrum of motor neuron diseases, not CGL2 [1,67]. These individuals do not suffer from fat metabolism disorder or abnormal distribution of body fat, but do suffer from many motor neuron diseases, such as Silver Syndrome (also known as spastic paraplegia type 17), distal hereditary motor neuropathy type V, and Charcot-Marie-Tooth type 2 [1,67], which are termed "seipinopathies" by Ito and Suzuki [104]. However, following the terminology used for diseases caused by lamin A/C (LMNA) gene mutations, all diseases caused by BSCL2 mutations (CGL2, PELD, and BSCL2associated motor neuron diseases) should be called "seipinopathies" [59,105]. Therefore, we call "seipinopathies termed by Ito" other than CGL2 and PELD as "BSCL2-associated motor neuron diseases" as per a dyadic nomenclature in this review. The main manifestations of patients with BSCL2-associated motor neuron diseases are distal limb muscle weakness and atrophy, hypertonia, abnormal reflex, gait abnormalities, and so on [67,71]. There is phenotypic heterogeneity observed in motor neuron damage caused by the different variant (heterozygous N88S or S90L mutation) of BSCL2 [106,107], but the muscle magnetic resonance imaging pattern is consistent in that thenar eminence, soleus, and tibialis anterior are most frequently involved [71]. Two distinct simple heterozygous (c.566T > A, c.445C > G) missense BSCL2 variants are associated with epileptic encephalopathy, which have been reported in three patients [105,108]. Furthermore, a single BSCL2 variant including p.N88T [109], p.S141A [109], or p.R96H [110] may also result in one patient with motor neuron diseases, respectively. Details of phenotypes caused by these mutations still warrant further study.

Treatment for Human Diseases Caused by BSCL2 Mutations
The clinical treatment for diseases caused by BSCL2 mutations is mainly directed towards symptoms [58], and we summarise studies that report specific drug therapy here (Table 3). In two CGL2 patients, it has been shown that pioglitazone improves lipodystrophy, insulin resistance, diabetes mellitus, and hypoleptinaemia [111,112]. In 30 CGL2 patients, leptin-replacement therapy improves metabolic disorders including diabetes mellitus, insulin resistance, hypertriglyceridaemia, fatty liver, and elevated plasma angiopoietin-like protein 3 [113][114][115][116][117][118]. Leptin-replacement therapy may be associated with normalising regular menses of female CGL2 patients. A 12-year-old female CGL2 patient who had not yet started her menses, did do so after receiving leptin. Another seven female patients between the ages of 15 and 40 years with lipodystrophy (5 patients with CGL1 and 2 patients with acquired generalised lipodystrophy) have commenced regular menses from primary amenorrhea or abnormal menstruation after this therapy [96]. Finally, in two PELD patients, leptin-replacement therapy delayed the neurological regression and allowed better seizure control [119,120].

Role of Seipin in CGL2
In 2011, Cui et al., established the first CGL2 animal model in mice, also named seipin knockout (SKO) mice, by Bscl2 ablation through homologous recombination in embryonic stem cells [121]. The SKO mice recapitulate most of the metabolic phenotypes of human CGL2 patients, including significant loss of adipose tissue mass, glucose intolerance, liver steatosis, and hyperinsulinaemia [121]. However, in contrast to CGL2-associated hypertriglyceridaemia, SKO mice have hypotriglyceridaemia. This hypotriglyceridaemia may be due to increased clearance of TAG-rich lipoproteins and uptake of fatty acids by the liver, with reduced basal energy expenditure in mice [121,122]. Cardiomyopathy [123] and renal injury [124] induced by metabolic disorders in SKO mice are also noted. Consistent with CGL2 patients, the SKO mice also experience abnormalities in their nervous and reproductive systems. The neurological symptoms of SKO mice are mainly as follows: anxiety [8], depression [8,125], spatial cognitive deficits [9], and impairment of motor coordination [10]. For reproductive system changes in SKO mice, the males have both teratozoospermia and complete infertility [14,15], while all females have accelerated mammary gland ductal growth but delayed vaginal opening [11], insufficient milk production [12], and defective parturition [126]. The above phenotypes of this CGL2 animal model were further validated in mice by using another gene-editing technology (CRISPR-Cas9) to delete Bscl2 in 2020 [127].
Since the expression pattern of seipin in the organs of rats is more similar to humans than in mice, Ebihara et al., established the first CGL2 rat model by using the method with ENU (N-ethyl-N-nitrosourea) mutagenesis for SKO in 2015 [128]. It has been found that the SKO rats are hypertriglyceridaemic, implying that it is a better model to represent blood lipid patterns in metabolic disorders observed in CGL2 patients [128]. The SKO rats also experience the impairment of memory abilities and infertility with azoospermia [128].

Role of Seipin in Metabolic Disorders
Metabolic disorders are the predominant characteristics of SKO mice and attract numerous researchers investigating the underlying mechanisms and potential therapeutic targets (Table 4). Magré and Prieur have elegantly summarised the phenotypes and underlying mechanisms of metabolic disorders and the complications of CGL2 in mice in a recent review [45]. Therefore, rather than replicating their findings, this review complements this by adding some other findings that are not mentioned in their review.
In the review, Magré and Prieur described that seipin deficiency causes severe lipodystrophy by impairing adipogenesis, accelerating lipolysis, damaging LD homeostasis and other unclear mechanisms [45]. Here, we want to discuss whether the metabolic disturbance is caused by adipose tissue loss in SKO mice. On the one hand, the following evidence supports the conclusion that the metabolic disorders are caused by seipin deficiency induced adipocyte tissue loss: (1) aP2-driven seipin deletion induced glucose intolerance, insulin resistance, and liver steatosis in 6-month-old mice [44]; (2) mice are more susceptible to high-fat-diet-induced insulin resistance and hepatic steatosis because of acquired Bscl2 deletion on mature adipose tissue [46]; (3) transplantation of normal adipose tissue ameliorates insulin resistance, dyslipidaemia, and severe hepatic steatosis in SKO mice [139]; (4) seipin deficiency results in obvious metabolic disturbance of liver or skeletal muscle in SKO mice in a non-cell autonomous way [51,52,140]. On the other hand, there are also some researchers who hold the opposite opinion: (1) there is no overt metabolic dysfunction in adiponectin-driven seipin deletion male mice, even following a high-fat diet challenge [46,136]; (2) adiponectin-driven seipin-deletion female mice fail to show severe metabolic disturbance, and only when facing the challenge of a high-fat diet at thermoneutrality do they develop moderate metabolic dysfunction [137].     Additionally, accumulating evidence also puts forward that seipin also participates in other physiological functions: (1) Seipin may play an important role in insulin synthesis and secretion via regulating the expression of PPARγ in pancreatic islets, and this function is affected by oestradiol [13]. (2) Seipin also plays a key role in plasma cholesterol metabolism together with low-density lipoprotein receptor (Ldlr). It has been found that an atherogenic diet induces much higher plasma cholesterol (6000 mg/dL) in seipin and Ldlr double knockout mice compared with SKO mice (300 mg/dL) or Ldlr knockout mice (1000 mg/dL) alone [133]. (3) Seipin contributes to normal cardiac function. It is well known that SKO mice develop hypertrophic cardiomyopathy with diastolic dysfunction [45]. Researchers have studied the underlying mechanisms of it in SKO mice and in cardiomyocyte-specific seipin knockout (seipin-cKO) mice. In SKO mice, hypertrophic cardiomyopathy is mainly caused by diabetes mellitus [45]. However, in seipin-cKO mice, dilated cardiomyopathy with systolic dysfunction is mainly induced by increasing ATGL expression, excessive fatty acid oxidation (FAO), as well as a drastic reduction in cardiac lipidome [144]. (4) Seipin plays an important role in vascular function. Wang et al., found that seipin deficiency leads to a reduction in perivascular adipose tissue mass and adipose-derived relaxing factors secretion, impairing vessel contractility and relaxation [142]. Bruder-Nascimento et al., proposed that hypoleptinaemia in SKO mice induces increased vascular adrenergic contractility and impairs endothelium-dependent relaxation via PPARγ-dependent mechanism increasing NADPH oxidase 1 expression and reactive oxygen species production [141,146]. Additionally, seipin deficiency induces hypertrophic vascular remodelling in which the underlying mechanism remain unclear [141].

Role of Seipin in Neurodegenerative Phenotypes
Seipin deficiency can result in neurodegenerative phenotypes through a variety of mechanisms, which can be summarised as the following aspects ( Figure 2 and Table 5): (1) Neuron-specific seipin knockout (seipin-nKO) induces neuron damage through inhibiting PPARγ and its downstream targets [8][9][10]20,125]. Studies in SKO, seipin-nKO, and seipin-aKO mice show that seipin deficiency in neurons (both SKO and seipin-nKO), but not adipose tissue, causes neurological injury by inhibiting PPARγ [8,9]. Since then, more and more studies have focused on how seipin deficiency induces neurodegeneration via inhibiting PPARγ. In hippocampal CA1 regions, PPARγ inhibition could selectively suppress α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) expression through decreasing the activity of extracellular signal-regulated kinase (ERK) and cAMPresponsive element binding protein (CREB), resulting in N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) and spatial memory impairment [9]. An in vitro study also finds knockdown of Bscl2 gene expression impairs excitatory postsynaptic currents via reducing levels of surface AMPAR [147]. In the hippocampal dentate gyrus, PPARγ inhibition not only impairs the proliferation of stem cells by inhibiting ERK phosphorylation but also inhibits the differentiation of progenitor cells, which is caused by the reduction in neurogenin 1 (Neurog1) and neurogenic differentiation 1 (NeuroD1) expression via inhibiting Wnt3 signal [125]. In hippocampal neurons, PPARγ inhibition induces hyperphosphorylation and aggregation of tau phosphorylation at Thr 212 /Ser 214 and Ser 202 /Thr 205 through increasing protein kinase B-mammalian target of rapamycin (AKT-mTOR) signalling, which not only suppresses autophagy, but also elevates the glycogen synthase kinase-3β (GSK3β) activity, eventually leading to axonal atrophy [20,148]. In dopaminergic neurons, PPARγ inhibition elevates activated GSK3β-induced α-synuclein phosphorylation and neuroinflammation, and decreased PPARγ caused α-synuclein aggregation, resulting in dopaminergic cells loss [10]. (2) Metabolic dysfunction caused by SKO mice also contributes to its neurodegenerative phenotypes. Insulin resistance is responsible for neuron injury through increasing tau phosphorylation at Ser 396 induced by the c-Jun N-terminal kinase (JNK) pathway [148], and low adipokine levels have been certified to be involved in the pathogenesis of neurovegetative disease because leptin plays a key role in memory performance via maintaining hippocampal function, and adiponectin takes part in various brain functions in physiology [149][150][151].
vates the glycogen synthase kinase-3β (GSK3β) activity, eventually leading to axonal atrophy [20,148]. In dopaminergic neurons, PPARγ inhibition elevates activated GSK3β-induced α-synuclein phosphorylation and neuroinflammation, and decreased PPARγ caused α-synuclein aggregation, resulting in dopaminergic cells loss [10]. (2) Metabolic dysfunction caused by SKO mice also contributes to its neurodegenerative phenotypes. Insulin resistance is responsible for neuron injury through increasing tau phosphorylation at Ser 396 induced by the c-Jun N-terminal kinase (JNK) pathway [148], and low adipokine levels have been certified to be involved in the pathogenesis of neurovegetative disease because leptin plays a key role in memory performance via maintaining hippocampal function, and adiponectin takes part in various brain functions in physiology [149][150][151].

Role of Seipin in Reproductive Phenotypes
In experimental animal models of BSCL2 mutations, the potential effects on the reproductive system are proposed (Table 6).

3-12 months old
The age-related deficit in motor coordination induced by dopaminergic neuron injury, which is caused by enhanced aggregation and phosphorylation of α-synuclein or neuroinflammation was found in seipin-nKO mice and SKO mice but not seipin-aKO mice.

20-24 weeks old
The phosphorylation and aggregation of tau protein in seipin-nKO mice and SKO mice were more severe than that in seipin-aKO mice. Phosphorylation and aggregation of tau protein at Thr 212 /Ser 214 and Ser 202 /Thr 205 were abolished through rosiglitazone or A014418 or rapamycin or LY294002 in seipin-nKO or SKO mice, and hyperphosphorylated tau at Ser 396 in SKO mice was corrected by rosiglitazone or SP600125.  Studies in SKO, germ-cell-specific seipin knockout (seipin-gKO), and seipin-aKO mice show that the absence of seipin protein in germ cells (SKO and seipin-gKO), but not in adipose tissue, leads to infertility and teratozoospermia [14]. The underlying reasons for seipin-gKO-induced phenotype could be concluded as follows: Lipid accumulation caused by altered phospholipid metabolism in seipin loss germ cells may contribute to the structural defects in spermiogenesis and severely abnormal sperm [14]. Seipin may play a key role in DNA protection because Bscl2 is strongly induced after exposure to genotoxic agents in mouse embryonic stem (mES) cells (but not in primary liver cells) [152]. Seipin deficiency increases chromocenter fragmentation and defective chromatin condensation in sperm cells, impairing sperm quantity and motility [15]. Additionally, in mice without seipin in germ cells, defective mitochondrial activity and resultant impaired sperm motility also contributes to infertility [15].
According to previous studies, phenotypes involving the female reproductive system induced in SKO mice mainly include abnormal mammary gland development, insufficient milk production, and defective parturition. Female SKO mice have longer and more dilated mammary gland ducts at 5 weeks old [11], and the downregulation of oestrogen receptor β in mammary gland may be responsible for this phenomenon because it may have an antiproliferative function as it does in the uterus [11,153,154]. All adipocytes, LDs, and seipin itself have a role in milk production. White adipocytes transform into milk-secreting epithelial cells during pregnancy and lactation [155]; seipin-involved formation of mature LDs is one step of milk production by mammary epithelial cells [4,156]. Seipin is not only one component of the milk lipid globule membrane [157], but it also protects mammary gland alveolar epithelial cells from ER stress to avoid poor differentiation and/or apoptosis to guarantee normal milk secretion [12]. As for defective parturition in female SKO mice, myometrial hypertrophy caused by metabolic disturbance and elevated autophagy induced by uterine luminal epithelium seipin loss could both be expected to be responsible for this [126].

Role of Different BSCL2 Variants in Motor Neuron Diseases
BSCL2-associated motor neuron diseases damage upper motor neurons, lower motor neurons, and peripheral motor axons differently [104]. To date, there are three animal models for BSCL2-associated motor neuron diseases, including N88S mutant seipin transgenic (N88S seipin Tg) mice [158], N88S seipin Tg zebrafish [159], and N88S/S90L mutant seipin transgenic (N88S/S90L seipin Tg) mice [160]. Both the N88S seipin Tg mice and N88S/S90L seipin Tg mice reproduce the most symptomatic phenotype of patients with BSCL2-associated motor neuron diseases, including the progressive spastic motor deficit, reactive gliosis in the spinal cord, and neurogenic muscular atrophy [158,160]. The main phenotype of N88S seipin Tg zebrafish is a reduction in spontaneous free swimming of zebrafish [159].
The potential molecular mechanisms are summarised as follows: (1) Whether ER stress should be considered as a predominant mechanism is a subject of debate. Mutations of N88S and S90L in Bscl2 inhibit the glycosylation of seipin, which leads to the misfolded and aggregated proteins and the excessive accumulation of misfolded proteins, resulting in ER stress in neurons [1,161,162]. N88S seipin causes ER stress in vitro, which is partially attributed to a reduction in TAG content [159]. In contrast, some researchers find that ER stress is not obvious in both N88S/S90L seipin Tg mice and N88S seipin Tg zebrafish [159,160]. Therefore, further work is required to ascertain the definitive role of ER stress in BSCL2-associated motor neuron diseases. (2) N88S/S90L seipin expression results in the formation of inclusion bodies in the cytoplasm. Compared with normal cells, N88S/S90L seipin induces the formation of inclusion bodies in the cytoplasm in different cell types, including motor neurons at a 25-30 percent level in vitro [161,162]. N88S seipin Tg mice and N88S/S90L seipin Tg mice also show that inclusion bodies are formed in the cytoplasm of neurons [158,160]. The formation of inclusion bodies may be an adaptive machinery against the accumulation of N88S/S90L seipin in ER and its associated ER stress [158]. However, there are only aggresomes, but not inclusion bodies, reported in patients [1]. Ito et al., found that inclusion bodies in experimental animals or cells are different from aggresomes in humans, because both their component proteins and formation locations are different [1,162,163]. (3) Seipin N88S expression affects the LD morphology and decreases the TAG content of nervous tissue. Many small and sometimes clustering LDs, similar to LDs caused by seipin deficiency, are induced by N88S/S90L seipin in vitro. The expression of seipin N88S leads to a reduction in TAG storage in both motor neuron cells and zebrafish [159,164]. Seipin maintains axonal function in long corticospinal neurons together with Reep1 through regulating LD formation and morphology [165]. Additionally, N88S/S90L seipin significantly activates the autophagy pathway in the central nervous system of mice, eventually leading to motor neuron death [160].

Research Advances Acquired from Diseases Associated with Abnormal Expression of Seipin
The role of seipin in diseases not caused by BSCL2 mutations has attracted more and more attention in recent years. At present, the relationships between seipin and metabolic [16], neurodegenerative [10,[17][18][19][20]166], and other diseases [21,167,168] have been reported.
It is proposed that some diseases not caused by BSCL2 mutations affect the levels of seipin expression, and the abnormal seipin expression would in turn affect disease progression (Table 7). In hepatic steatosis, seipin expression is downregulated, which promotes intracellular TAG storage via calcium-depletion-induced ER stress, and adenoassociated virus-mediated seipin overexpression in liver suppresses intracellular TAG accumulation via increasing cytosolic calcium [16]. In patients with Parkinson's disease, the proteomic analysis of substantia nigra shows that expression of seipin is decreased [17]. Intriguingly, seipin expression levels are significantly increased in experimental Parkinson's disease models both in vitro and in vivo, and overexpression of seipin aggravates ER stress via the Grp94/Bip-ATF4-CHOP signalling pathway [166]. Seipin deficiency facilitated Aβ 25-35/1-42 -induced neuroinflammation via the PPARγ-GSK3β-TNF-α/IL-6 pathway in astrocytes, resulting in neurodegenerative disorders eventually [20,169]. In glioblastoma patients and glioblastoma cell lines, the expression of BSCL2 is increased [167]. In ischaemia/reperfusion-induced cerebral damage, the expression of seipin is inhibited by elevated miR-187-3p (a microRNA which can bind to the protein-coding sequence of seipin), and suppression of seipin expression increases neuronal apoptosis via deficient autophagic flux and ER stress [18,19]. Furthermore, SKO mice have exacerbated neurological disorder and enlarged infarct size that may be caused by increased blood-brain barrier permeability, amplified ER stress, and elevated glucose levels, as well as decreased leptin and adiponectin levels [170]. In a cardiac hypertrophy model in mice, seipin deficiency accelerates diastolic heart failure after transverse aortic constriction because of abnormal myocardial calcium handling and enhanced ER stress [21]. Recently, researchers have also found that the expression of seipin is inhibited in the myocardium of an intrauterine growth restriction sheep model [168].  Seipin deficiency worsened cardiac hypertrophy and diastolic heart failure which may be related to the impairment of myocardial calcium handling, ER stress, inflammation, and apoptosis in the heart failure model.

Lessons Learnt from Experimental Animals: Seipin as Therapeutic Target
To date, the main treatments for diseases caused by BSCL2 mutations are directed towards symptoms without specific and targeted therapeutic options [58,59], and yet, research on targeted therapy for PELD and BSCL2-associated motor neuron diseases is limited. Here, we summarise that seipin deficiency causes phenotypes via inducing adipose tissue loss or impairing the seipin signalling pathway, and two main strategies can be proposed to treat CGL2: (1) restoration of adipose tissue itself or its function to treat metabolic complications resulting from adipocyte seipin deficiency; (2) tackling seipin or seipin upstream or downstream targets to alleviate the effects of seipin deficiency in organs in which seipin works in a cell-autonomous way.
The restoration of adipose tissue function, which includes improving TAG storage capacity and maintaining adipokine levels, is the main principle to treat metabolic disorders and complications in CGL2 [171]. The potential modalities for restoring adipose tissue function can be concluded as follows: (1) normal adipose tissue transplantation; (2) PPARγ agonists; (3) exogenous replacement for adipokines; (4) inhibition of GPAT3 or ATGL; (5) restoration of seipin expression in adipose tissue. Details are as follows: (1) In SKO mice, metabolic disturbance and its complications, including insulin resistance, severe hypoleptinaemia, and severe hepatic steatosis, as well as renal injury, are abolished by adipose tissue transplantation [124,139]. (2) In SKO mice, pioglitazone ameliorates insulin resistance [122], liver steatosis [122], and hypertrophic cardiomyopathy [135]. In comparison with pioglitazone, rosiglitazone treatment not only rescues metabolic disorders in SKO mice [13,44], but also alleviates hyperlipidaemia and atherosclerosis caused by simultaneous absence of seipin and Ldlr in mice [133]. However, in SKO rats, both pioglitazone and rosiglitazone fail to rescue the fatty liver via rescuing PPARγ expression in the liver and adipocyte tissue. Therefore, whether the potential that fatty liver could be rescued by PPARγ agonists is species-dependent or related to the methodology in which SKO animals are generated remains unclear [145]. (3) In SKO mice, leptin-replacement therapy not only rescues impaired vascular function [141,146], but also ameliorates kidney injury secondary to metabolic disturbance [124]. In parallel, fibroblast growth factor 21, another adipokine, has also been used to improve the metabolic profile of white adipose tissue to enhance insulin sensitivity and increase plasma adiponectin levels in SKO mice [134]. (4) GPAT3 ablation significantly improves insulin sensitivity and liver steatosis in SKO mice through increasing adipocyte tissue mass [127]. Similar to GPAT3, adipose tissue mass is tightly correlated with ATGL expression in SKO mice [48], and heterozygous deletion of ATGL significantly ameliorates metabolic disorders and cardiac hypertrophy; complete ablation of ATGL even fully reverses seipin deficiency-induced lipodystrophy [48]. (5) In SKO mice, the recovery of seipin expression in adipocyte tissue improves metabolic disturbance [131].
In organs where seipin works in a cell-autonomous way, tackling seipin-related PPARγ pathways is the main principle to ameliorate disorders of these organs in CGL2. In SKO mice, rosiglitazone treatment ameliorates anxiety [8], depression [8,125], spatial cognitive impairment [9], and motor impairment [10], as well as Aβ 25-35/1-42 -induced Alzheimer's disease [20]. In seipin-nKO male mice, either rosiglitazone or oestradiol ameliorate anxiety and depression via rescuing PPARγ [8]. Rosiglitazone or oestradiol also work in rescuing pancreatic function in male mice with heterozygous deletion of seipin [13]. The downstream targets of PPARγ also show great therapeutic potential: a mitogen-activated protein kinase kinase (MEK) inhibitor blocks the rosiglitazone-rescued depression and spatial memory in seipin-nKO mice [9,125]; a GSK3β inhibitor ameliorates α-synuclein phosphorylation, neuroinflammation and the hyperphosphorylation and aggregation of tau protein in seipin-nKO mice [10,148]; and a phosphoinositide 3-kinase inhibitor or mTOR inhibitor accelerates p-tau and tau protein clearance, relieving axonal atrophy in the hippocampal neurons of seipin-nKO mice [148].
In addition, some treatments that cannot be concluded by these two strategies also ameliorate CGL2 phenotypes. In SKO mice, the sodium-glucose cotransporter 2 inhibitor dapagliflozin successfully reduces the O-GlcNAcylated protein levels and blocks the de-velopment of hypertrophic cardiomyopathy [135]; JNK inhibitor improves hippocampal neuron damage through reducing hyperphosphorylated tau at Ser396 induced by the insulin resistance cascading JNK pathway [148]; miR-187-3p inhibitor ameliorates ischemiainduced cerebral damage significantly by rescuing seipin expression to improve autophagic flux and relieve ER stress [18,19]; and genistein treatment accelerates vaginal opening but increases mammary gland area in SKO mice [11].

Conclusions and Perspective
In the current review, the summarised evidence suggests that seipin plays an important role not only in rare diseases induced by BSCL2 mutations, but also in more common diseases that are not related to BSCL2 mutations. Limitations exist in studies focusing on the relationships between seipin and diseases: (1) studies on differential expression patterns of seipin in different diseases and the underlying mechanisms require exploration; (2) the precise mechanism of seipin in the brain and testis where it works in a cell-autonomous way is still unclear; and (3) there are rare seipin signalling-pathway-based treatment options for relevant diseases. Therefore, further studies to address these limitations are awaited. Moreover, similar to seipin, other ER-located proteins involved in LD biogenesis such as fat storage-inducing FIT2, Atlastin, REEP1 and so on are also closely associated with human diseases. For example, FIT2 has been certified to play a key role in lipodystrophy [172], diabetes mellitus [173,174], and hepatocellular carcinoma [175], as well as doxorubicinmediated cardiotoxicity [176], and the underlying mechanisms are also incompletely clear. Therefore, it is of great significance to study the precious mechanism of these proteins in diseases, and studies focusing on seipin are excellent examples of them.

Conflicts of Interest:
The authors declare no conflict of interest.