The Role of Saposins in Auditory and Vestibular Systems

Abstract Prosaposin, and its cleaved byproducts saposin A-D, are Akil O., Rouse SL., Lustig LR. crucial proteins necessary for glycoshpingolipid degradation and metabolism. Mutations in these proteins Omar Akil, PhD can lead to devastating lysosomal storage diseases, Department of Otolaryngology- including Gaucher’s disease, symptoms of which often

Head & Neck Surgery include hearing loss. Until recently, little was known about University of California San Francisco. the role that prosaposin and saposins A-D played in Email: [email protected] auditory and vestibular system function. Early studies in knockout mouse models showed prosaposin to be Stephanie L. Rouse, BA important for normal cochlear innervation and Department of Otolaryngology- maintenance of normal hearing (Akil et al., 2006). Severe Head & Neck Surgery vestibular dysfunction paired with inclusion body University of California San accumulation in the vestibular end-organs of prosaposin Francisco. KO mice suggests its role in the maintenance of normal Email: hearing and the maturation of normal vestibular system [email protected] function (Akil et al., 2012). To determine if these phenotypes were attributable to the loss of prosaposin as Lawrence R. Lustig, MD a whole or one of its constituent proteins, saposin A-D, KO Department of Otolaryngology- mice of individual saposins were subsequently studied. Head & Neck Surgery Given the nature of the hearing loss, as well as efferent Columbia University Medical and afferent neuronal sprouting in the prosaposin KO Center. Email: mouse, it was hypothesized that saposin C, a protein [email protected] known for its neurotigenic properties, was responsible for these changes. However, a null-mutant mouse lacking both saposin C and D showed no effect on hearing (Lustig Corresponding author: Omar Akil PhD et al., 2015). In contrast, a loss of functional saposin B led Department of Otolaryngology- to a progressive sulfatide accumulation in satellite cells Head & Neck Surgery, around cochlear spiral ganglion (SG) neurons resulting in University of California San satellite cell degeneration, SG degeneration, and Francisco, 533 Parnassus ultimately, loss of hearing (Akil et al., 2015). While saposin Avenue Room U490 San B KO mice did not show any vestibular dysfunction Francisco, CA, 94143-0449. phenotype, vestibular evoked potentials demonstrated Email: [email protected] profound vestibular dysfunction likely attributable to the large-scale neuronal degeneration (Akil et al., 2015). Phone: (01) 415-502-4880 Furthermore, the data suggests that saposin B appears to Fax: (01) 415-502-4880 have a much greater role in auditory neuronal maintenance and balance than saposin C and/or D.

Key Words: prosaposin, saposin A, B, C, D, glycosphingolipid hydrolases, cochlea, hearing, balance, vestibular, auditory

1. Introduction only serve as structural components of cell Prosaposin and its cleaved byproducts membranes, but it is now recognized that saposin A-D are crucial proteins necessary they are involved in signaling cell fate as for glycoshpingolipid (GSL) degradation, well as neuronal signaling (reviewed by mutations of which can lead to devastating Colombaioni et al., 2004). Sphingolipd lysosomal storage diseases such as metabolites have a clear role as signal Gaucher’s disease. These proteins are transducers involved in cell differentiation, important targets of study as they offer migration, growth, apoptosis, proliferation, potential treatment for such disorders as and survival (Colombaioni et al., 2004). well as offer insight into neuronal Clinically they are important - genetic and pathfinding. Until recently, little was known metabolic abnormalities of these about prosaposin or saposins’ role in the compounds result in a number of diseases ear but many of these lysosomal storage with a variety of neuronal deficits, including diseases include symptoms of progressive Fabry disease, Krabbe disease, hearing loss in patients, and thus draw metachromatic leukodystrophy, Niemann- interest to the ear. Here we review what is Pick disease, and Tay-Sachs disease known about prosaposin and each saposin (Sandhoff et al., 1970). (A-D) constituent as they relate to auditory and vestibular function. Sphingolipid activator proteins, termed saposins, are four small glycoproteins all generated in lysosomes from a single 1.1 Sphingolipid Overview Sphingolipids, also known as precursor protein, prosaposin (Kishimoto glycosylceramides, are a class of lipids, made et al., 1992). Each of the four saposins A-D up of different combinations of an amide- are necessary for activity of specific linked fatty acid and a long-chain (sphingoid) lysosomal glycosphingolipid hydrolases base, that were discovered in 1870 by Johann (GSLs) involved in the metabolism of Thudicum when examining brain extracts. various sphingolipids (Sun et al., 2002). Thudicum deemed the enigmatic chemical These proteins are similarly important as structure “sphinx-like” and so named the individual saposin mutations are molecules ‘sphingosin’ (reviewed by associated with lysosomal storage Hakomori, 1983). The biochemistry of these diseases, some associated with progressive and related compounds are based on a hearing loss such as Gaucher’s disease ceramide backbone. Ceramides are (Holtschmidet et al., 1991; Paton et al., sphingolipids with an R group consisting 1992; Bamiou et al., 2001; Campbell et al., solely of a hydrogen atom. Sphingomyelin is 2004). similar to a ceramide but the R group is phosphocholine. If the R group is a sugar 1.2 Prosaposin Overview monomer, the compound is a cerebroside, Prosaposin is a multifunctional protein (70kDa) encoded by a single gene on and if it is a sugar polymer it is termed a chromosome 10 with extra and intra globoside; together these two compounds are cellular functions (Inui et al., 1985; O’Brien known as glycosphingolipds (GSLs). et al., 1988; Fujita et al., 1996). Intracellularly, prosaposin is cleaved into As a group, the sphingolipids are critical for constituent lysosomal glycoproteins saposin neuronal signal transmission and cell A-D that degrade specific glycosphingolipids recognition. Initially they were believed to through activation of GSL hydrolases. Copyright 2015 KEI Journals. All Rights Reserved Page | 2

Medical Research Archives

Prosaposin moves from the endoplasmic proteases (O’Brien et al, 1991). The four reticulum to the Golgi and then the saposins are structurally similar to each endolysosomal compartment where it is other, yet the specificity and mode of proteolytically processed into four activation of sphingolipid hydrolases differs. homologous saposins of roughly 80 amino acids each (Sandhoff et al., 1998). The first saposin SAP-1, later termed Extracellularly, prosaposin is involved in lipid transfer and nerve outgrowth saposin B (SapB), was discovered by properties (O’Brien et al., 1995). Jatzkewitz et al in 1964. Research analyzing enzymatic sphingolipid degradation show-

ed water-soluble lysosomal hydrolases Originally named SGP-1, prosaposin was cleave their sphingolipid substrates first isolated in rat then detected in various effectively only in the presence of the right human tissues, primarily the brain and detergents (Sandhoff K, 1970; Scholte et al., testes followed by kidney, spleen, and liver 1991). Jatzkewitz et al. were searching for (O-Brien et al., 1988). Hineno et al. detected in vivo factors that substitute for detergents uncleaved prosaposin, in various human when they purified arylsulfatase A and body fluids, such as cerebrospinal fluid, discovered SapB. SapB is a small, unspecific seminal plasma, milk, pancreatic juice, glycoprotein shown to activate hydrolysis blood plasma, and bile, suggesting several of cerebroside sulfate by arysulfatase A and functions of prosaposin (Hineno et al., activate hydrolysis of ganglioside G1 (GM1) 1991). Prosaposin and its constituent by acid β-galactosidease (Sandhoff et al., saposins are widely expressed in many 2012). As such, SapB is a physiological tissues and can be considered detergent that facilitates the partial “housekeeping proteins” necessary for extraction of GSLs from membranes and lysosomal hydrolysis (Radin et al., 1984). As presents them to the enzymes arylsulfatase such, most studies on prosaposin A and α-galactosidase A (Sandhoff, 1992). In expression have been done in the brain, kidney, and liver with little or no focus in humans, SapB inherited deficiency causes the progression of metachromatic the ear. A null mutation of the prosaposin gene in mice leads to a complex leukodystropy-like disease caused by neurodegenerative phenotype as well as arylsulfatase A deficiency (Zhang, 1990). severe leukodystrophy and abnormally increased levels of brain gangliosides The second saposin SAP-2, now called (Fujita et al., 1996). saposin C (SapC), was discovered by Ho and O’Brien in 1971 and found to increase activity of the lysosomal enzyme β- 1.3 Saposin A-D Overview Saposins A-D are vital for sphingolipid and glucosidase. Its deficiency leads to a membrane degradation at inner lysosomal Gaucher-like disease with neurological membranes. They bind lipids and transfer manifestations due to decreased them as water-soluble complexes from glucosylceramide metabolism (Zhang, vesicular membranes to catabolizing 1990). enzymes or acceptor membranes. Each saposin has six conserved cysteine residues As more saposins were identified, the forming disulfide bonds, two prolines, as nomenclature changed based on the well as an N-glycosylation site and so are placement of the four saposin domains in incredibly heat stable and resist most prosaposin, reading from amino- to Copyright 2015 KEI Journals. All Rights Reserved Page | 3

Medical Research Archives carboxyl-terminal, saposins A, B, C, and D tissues where sphingolipids accumulate. (Morimoto et al., 1988). This change cleared Much work has been done to explore up confusion surrounding the naming of saposins in these organs but it was not until SAPs across species and emphasized their hearing loss phenotypes associated with common genetic origin. these diseases were observed that research was done into saposins’ role in auditory Saposins A and D were discovered in 1989 function. In one study of patients with and 1988 from cloning and sequencing Gaucher’s disease, related to a saposin C- prosaposin cDNA (Morimoto et al., 1988; like deficiency, hearing loss was felt to have 1989). Less is known about these proteins. been attributable to an efferent auditory Fabbro and Grabowsky found saposin A defect; two-thirds of the patients in the (SapA) to stimulate glucocerebrosidase, and study had abnormal contralateral thus its function appears similar to SapC suppression of otoacoustic emissions (Fabbro et al., 1991; reviewed by Kishimoto (Bamiou et al., 2001). Despite these et al., 1992). While no patients with SapA associations, not much is known about deficiency have been identified, SapA saposins’ role in auditory and vestibular knockout (KO) mouse models exhibit systems. In this review we describe the similar pathology to that seen in chronic current understanding of the precursor form of globoid cell leukodystrophy and glycolipid, prosaposin, and its four lysosomal storage disorder Krabbe disease constituent’s role in the auditory and (Matsuda, 2004). vestibular systems.

Saposin D (SapD) is the least understood of 2. Prosaposin the saposins. Located near the C-terminus of prosaposin, SapD stimulates 2.1 Prosaposin in Auditory Function sphingomyelinase activity. SapD deficiency, Initial work describing prosaposin in the in both mice and humans, has been tied to auditory system identified the protein in the cerebellar Purkinje cell degeneration. ear via a yeast-two-hybrid screen, traced its Mutations in SapD have also been tied to expression in the ear over time, and urinary system defects manifested as phenotypically characterized prosaposin KO polyuria and polydipsia (Azuma, 1994; mice (Akil et al., 2006). This work not only Matsuda, 2004). SapD null mutation led to connected prosaposin to the auditory urinary and cerebellar deficits, yet a system but also identified prosaposin’s role combined saposin CD KO mouse led to in maintaining normal innervation patterns changes in prosaposin trafficking and to the organ of Corti. ceramide accumulation (Matsuda, 2004).

Saposin research was prioritized when it Akil et al. were screening for protein- protein interactions involved in the rodent was observed that patients with mutations efferent auditory system when they in the prosaposin gene developed lysosomal identified prosaposin. The authors found storage diseases accompanied by an diffuse expression of prosaposin in the accumulation of sphingolipidosis in many organ of Corti at birth, gradually localizing tissues (Kishimoto et al., 1992). The liver to supporting cells, Deiters’ cells, inner hair and brain of patients with Niemann-Pick, cells (IHCs), inner pillar cells, and the Tay-Sachs, Sandhoff, and Gaucher diseases synaptic region of outer hair cells (OHCs) by all show considerable excess of saposins in post-natal day 21 (P21). Additionally, Copyright 2015 KEI Journals. All Rights Reserved Page | 4

Medical Research Archives quantitative polymerase chain reaction (q- one of its derivatives was found to be critical PCR) and immunohistology experiments on for competent neuronal path finding and microdissected OHCs and Deiters’ cells subsequent maintenance of normal hearing. localized prosaposin mRNA to both Observations of prosaposin KO mice behavior populations, and found that protein was during auditory work led Akil et al. to predominantly localized to the apex of the question if prosaposin had a similar role in Deiters’ cells. the vestibular system.

Further work in prosaposin KO mice demonstrated a slight reduction in hearing in 2.2 Prosaposin in the Vestibular System mice up to P19 and deafness by P25 During these early studies on prosaposin in accompanied by reduced distortion product the cochlea, it was noted that prosaposin KO otoacoustic emissions from P15 onward (Akil mice demonstrated behaviors consistent et al 2006) Simultaneously, beginning at P12, with vestibular dysfunction, including the prosaposin KO mice showed histologic circling, an unsteady gait, and difficulties in organ of Corti changes including cellular maintaining balance. These observations hypertrophy in the region of the IHCs and suggest that in addition to its role in greater epithelial ridge, a loss of OHCs from hearing, prosaposin also contribute in cochlear apex, and vacuolization of OHCs. maintaining vestibular function. The Immunofluorescence revealed exuberant vestibular system is made up of three end- overgrowth of auditory afferent neurites in organs: the semicircular canals that detect the region of the IHCs and proliferation of changes in angular acceleration; the utricle auditory efferent neurites in the region of the that detects changes in linear acceleration; tunnel of Corti. IHC electrophysiologic and the saccule that detects changes in head recordings from these KO mice showed position with respect to gravity (Walls, normal current-voltage curves and responses 1962; Land, 1999; Spoor et al., 2002). In to applied acetylcholine, indicative of normal normal wild-type (WT) mice, prosaposin hair cell function. This work was supported localizes to the three vestibular end-organs by later work from Terashita et al (2007) and Scarpa’s ganglion as shown by RT-PCR, who also demonstrated localization of Western blot analysis, and prosaposin within the rat cochlea. Taken immunofluorescence (Akil et al., 2012). together, these results strongly suggest that prosaposin or one of its cleaved products is Ablation of prosaposin function caused severe required for maintenance of adult cochlear vestibular dysfunction on a series of innervation patterns in the organ of Corti, behavioral tasks. Histologically, the KO mice and as a result the maintenance of normal demonstrated an exuberant cellular hearing (Akil et al., 2006). Clinically, these proliferation below the vestibular hair cells finding could potentially contribute to the with disruption of the supporting cells. effort to improve neuronal survival for Electron microscopy further demonstrated profoundly deaf patients who may be inclusion bodies and cellular proliferation cochlear implant candidates as improved disturbing the normal neuroepithelial neuronal survival may improve cochlear structure of the vestibular end-organs, implant performance (Gao, 1998; Marzella reminiscent to what was seen in the organ and Clark, 1999; Shepherd and Hardie, 2001; of Corti (Akil et al., 2006). Nakaizumi et al., 2004). Prosaposin and/or Immunofluorescence staining with anit-

Medical Research Archives neurofilament F200 and anti-synaptophysin properties and stimulates the degradation antibodies in the vestibular tissues sections of sulfatide, a major lipid component of suggested that the cellular proliferation myelin, as well as other glycolipids (Li et al., corresponds to afferent and efferent 1988; Ciaffoni et al., 2006). SapB facilitates neuronal overgrowth, similar to what was partial extraction of target GSLs from seen in the organ of Corti (Akil et al., 2006). membranes, including sulfatide and From these studies it was concluded that globotriaosylceramide, forming soluble prosaposin played a role not only in the protein-lipid complexes and presenting maintenance of normal hearing, but was them for cleavage to arylsulfatase A (ASA) also critical in the neuronal maturation and α-galactosidase A, respectively processes of the vestibular sensory (Sandhoff et al., 1992; Furst et al., 1992). In epithelium and the maintenance of normal humans, Sap B deficiency leads to elevated vestibular system function. sulfatide levels in the kidneys and brain, variable neurological phenotypes and a However, previous work done in prosaposin metachromatic leukodystrophy-like disease KO mice, which demonstrated a number of similar to that observed with the deficiency abnormalities within the central nervous of ASA (O’Brian et al., 1995; Gieselmann et system, must be considered while al., 2010; de Hossen et al., 2011). interpreting these findings. These studies found similar pathologic changes as seen in Metachromatic leukodystrophy (MLD) is an the inner ear, including an inclusion body autosomal recessive neuro degener-ative accumulation with leukodystrophy and lysosomal disease caused by defective ASA gylcosphingolipid accumulation (Fujita et activity. MLD variants are also characterized al., 1996; Sun et al., 2002). As a result, it is by accumulation of sulfatides, progressive possible that a central pathology is a key demyelination, and extensive white matter contributor to the balance dysfunction damage (Hess et al., 1996). Like the human observed in KO mice. Perhaps it is a general disease, transgenic mice with diminished ASA central nervous system phenotype observed enzymatic activity develop accumulation of in prosaposin KO mice as opposed to sulfatide in white matter, and an early onset vestibular dysfunction, or a combination of neurological phenotype without evidence of both. demyelination (Hess et al., 1996). These ASA- deficient mice were also noted to be deaf, These initial studies raised the question lacking auditory brainstem response (ABR). whether the severe changes seen in organ of Several cases of MLD-like diseases caused Corti and vestibular end-organs were due to by Sap B deficiency have also been reported the loss of prosaposin itself or one of its with lysosomal storage of cerebroside cleaved end products, Saposin A-D. This sulfate (Wenger et al., 1989; Scholte et al., question motivated additional studies 1991). The Sap B defects in these cases arise exploring the roles of individual saposins in from various mutations that produce maintaining auditory and vestibular substitutions for critical cysteine residues, function. insertions, transversions or obliteration of a

glycosylation site (Sandhoff et al., 1992; 3. Saposin B

Saposin B (SapB) is a non-enzymatic GSL Deconinkik et al., 2008). activator protein that has lipid transfer Recently, Sap B deficient mice (B-/-) were developed by introducing a point mutation Copyright 2015 KEI Journals. All Rights Reserved Page | 6

Medical Research Archives into its coding sequence within prosaposin, Rosenthal canal, and modiolus, which destroying one of its three essential normally harbor the dendritic and neurotic disulfide bonds (Sun et al., 2008). This processes of the SG neurons, were almost mutation leads to an unstable and completely devoid of SG neurons and undetectable Sap B protein, but preserved myelinated nerve fibers. In contrast, the prosaposin and saposin A, C and D proteins overall architecture of the organ of Corti with their related functions. These B-/- mice and the stria vascularis were structurally mimicked the biochemistry and phenotype unaltered in the B-/-mice. The pattern of of the human disease and developed SG alteration resembled the one found in neurological impairment, including ataxia, ASA-deficient mice (Hess et al., 1996). head tremor, and impaired neuromotor Histological study of the SG neurons coordination (Sandhoff et al., 2001). At the revealed a progressive accumulation of microscopic level, neuronal storage of inclusion bodies in the satellite cells sulfatide in the auditory nerve was also surrounding the SG cells starting from identified (Sun et al., 2008). P1mo onwards. The degree of storage accumulation in the satellite cells increased 3.1 Saposin B in Auditory Function in older mice, which may have been the Based on prior work in ASA-deficient mice cause of SG degeneration that took place that exhibited hearing loss (Hess, 1996), between P4mo and P15mo in the B-/- mice. additional studies were performed by Akil However, the most severe alterations of SG et al. to evaluate cochlear changes in SapB neurons occurred between 4 and 8 months deficient mice to elucidate its role in hearing of age in the ASA-deficient mice (D’Hooge (Akil et al., 2015). Histological analysis of B- et al., 1999), indicating that in the B-/- /- mice revealed normal organ of Corti mice, there was a milder and slower morphology as compared to WT mice but a neurological and neuropathological significant reduction of spiral ganglion (SG) pheonotype to that observed in the ear of and cochlear nerve fibers in Rosenthal’s the ASA (-/-) mice (Coenen et al., 2001). canals that progressed with age. As opposed Similar observations were also seen for to prosasposin KO mice, neuronal other organs in the B-/- mice (Sun et al., overgrowth was not observed. Further 2008). The exact reason for this difference work showed that while gross morphology between the B-/- mice and the ASA- of the SG was identical to WT mice, there deficient mice is unknown. It is possible were abnormal inclusions in B-/- satellite that a lack of alternative ASA-independent cells highly correlated to loss of SG neurons pathways for sulfatide metabolism is that worsened with age. The inclusion responsible for the difference (Sundaram bodies in B-/- mice satellite and SG cells et al., 1995). It is also possible that, despite were determined to be sulfatide undetectable levels of Sap B, the mouse accumulation (Akil et al., 2015). model still has residual SapB function based on the nature of the mutation. These findings in the SapB KO mouse shed Alternatively, SapB deficiency may lead to important light on the role of saposins in accumulation of fewer GSLs than in ASA- auditory function. The histological results deficient mice, and hence a more mild from these data indicate a progressive phenotype (Akil et al., 2015). degeneration of SG cells and nerve fibers in the B-/- cochlea. The osseous spiral lamina, In addition to characterizing the histological Copyright 2015 KEI Journals. All Rights Reserved Page | 7

Medical Research Archives changes in the organ of Corti in SapB KO the afferent neuronal system to loss of Sap mice, auditory physiology was also studied B, and/or efferent neurons employ using auditory brainstem response (ABR) alternative metabolic pathways for GSL testing (Akil et al. 2015). No significant metabolism that allowed them to be spared differences in hearing between the B-/- the changes seen with the afferent system mice, heterozygote and WT mice were (Akil et al., 2015). This idea is supported by observed until P6mo. However beyond 6 findings that the dorsal root ganglia in B-/- months, a significant increase of ABR mice and other Saposin KO mice have thresholds were observed for each of the inclusions, but motor neurons are generally pure tone-burst stimuli (8, 16 and 32 kHz), not affected by these changes (Sun et al., but not click stimuli, in the B-/- mice. 2007, 2008) these results suggest Increases in ABR click stimuli thresholds in alternative manifestations of Sap function the B-/- mouse were not seen until P13mo. in afferent versus efferent neurons. It thus No defect in OHC function was observed in seems likely that OHC function was B-/- mice through P8mo despite 57% of SG preserved because there is no accumulation being lost. of sulfatide in the type 2 SG neurons.

It is interesting to note that ABR click 3.2 Saposin B in Vestibular Function stimuli thresholds were normal until Sun et al. reported that the B-/- mice P13mo, despite the severe morphologic demonstrate head tremor that may have changes seen in the spiral ganglion neurons been caused by the vestibular dysfunction developing during this same time period. and an abundant storage material Similar results have been documented in accumulation in the vestibular ganglion. mice models of hearing recovery after noise However, Akil et al. did not find any clear exposure, in which even ‘‘reversible’’ noise evidence for peripheral vestibular exposure with recovery of auditory dysfunction in B-/- mice. No head bobbing thresholds leads to long-term afferent nerve phenotype in the B-/- mice was observed terminal degeneration while retaining and the mutants were indistinguishable ‘‘normal’’ ABR thresholds (Kujawa et al., from the WT siblings even at older ages. 2009; Lin et al., 2011). Despite the abundant storage material accumulation in the vestibular ganglion, the These results imply a causative effect of Sap B-/-mice presented no visible signs of B deficiency on sphingolipid accumulation balance dysfunction. While B-/- mice did in inclusion bodies leading to neurological not show any vestibular dysfunction degradation and reduced hearing. The phenotype, vestibular evoked potentials observed pattern of hearing loss in both (VsEPs) demonstrated profound vestibular ASA-deficient and B-/- mice reflects the dysfunction likely attributable to large-scale morphologic changes observed (Akil et al., neuronal degeneration in myelinated SG 2015). ASA-deficient mice showed greater neurons. Because type 2 SG neurons are ABR threshold shifts at an earlier age, spared degeneration, the efferent system correlating with deterioration of the SG was preserved. The normal observed neurons and nerve fibers (D’Hooge et al., balance, despite the neuronal loss, is 1999). The intact efferent response is reminiscent of the normal click ABR suggestive of several possibilities. The thresholds observed in the same mouse efferent system may be more robust than where only 15% of the cochlea nerve fibers

Medical Research Archives remain at P15mo. This implies that not all Matsuda et al., 2004; Akil et al., 2006). In vestibular ganglion neurons are required both models, mutations were made in the for normal vestibular function or coding region to eliminate one of the alternatively the vestibular system has protein’s three essential disulfide bridges, more compensatory systems. Unlike creating a deficiency selectively in only the hearing, balance requires the integration of targeted saposin (Akil et al., 2006). several organ systems including vision and the musculoskeletal system, and it is When comparing mutant mice lacking either possible that these other systems offset the SapC or SapCD to WT littermates, loss of vestibular function, leading to a lack investigators found minimal histologic of an observable phenotypic change in changes in the organ of Corti and no effect on balance. hearing. Morphology of inner and outer hair cells, supporting cells, stria vascularis, and spiral ganglion neurons were all normal 4. Saposin C and Saposin CD through P30 in both the SapC and SapCD KO mice (Lustig et al., 2015). Additionally, 4.1 Saposin C and Saposin CD in the surface preparations stained with phalloidin auditory system showed normal inner and outer hair cell It was unclear from early studies of counts throughout the cochlea. As compared prosaposin if the severe inner ear deficits to WT littermates, neither mutation resulted were due to actions of prosaposin itself, in notable changes in ABR thresholds, and known to have intrinsic nerve-growth- both mutants exhibited normal outer hair factor like qualities (Furst et al., 1992; Sun cell function, as seen through DPOAE, et al., 2002). In a further attempt to demonstrating normal afferent and efferent determine if the severe inner ear deficits auditory neuronal function in the KO mice observed in prosaposin KO mice were due through P55. These results suggest that the to a lack of prosaposin or individual loss of SapC or SapCD has no observable constituents of prosaposin, investigators effect on the organ of Corti through this age. turned to examining the role of saposin C (SapC) in the ear. As SapC is known for 4.2 Saposin C and Saposin CD in the having neurotigenic properties, it was vestibular system hypothesized that SapC may be responsible While auditory function appeared normal, for the hearing loss and efferent and SapCD KO mice exhibited difficulty walking afferent neuronal sprouting seen in the and generally abnormal movement, prosaposin KO mice (Lustig et al., 2015). bringing the vestibular system into Given the neuritogenic and nerve question. The vestibular morphology of outgrowth properties of the n-terminal SapCD KO mice was similar but not as region of SapC it seemed likely that SapC severe as it was for prosaposin KO mice. was behind the neuronal proliferation Lustig group found that inclusion bodies observed in the prosaposin KO mice. To form in SapCD KO mice in all three end- explore this idea, two saposin transgenic organs and upon immunofluorescence mice were generated; one lacking SapC and staining for afferent and efferent auditory the other combining the null mutation of fibers; these cellular proliferations were saposin C and D to generate a saposin CD found to be consistent with neuronal (SapCD) KO mouse (Bamiou et al., 2001; overgrowth. The abnormal movement Copyright 2015 KEI Journals. All Rights Reserved Page | 9

Medical Research Archives patterns and inclusion bodies were not seen and dorsal root ganglion. These mice in the SapC KO mice nor in WT littermates, similarly showed large accumulations of are suggesting a lack of SapD responsible glucosylceramides and alpha-hydroxy for these inclusions. ceramides in their brains and kidneys. Thus, while there are abnormalities noted within To explore if the inner ear deficits found in the vestibular system in the SapCD KO mice, the prosaposin KO mouse were due to an they appear mild in comparison to the absence of prosaposin itself or if one of the central changes seen by Sun et al. end-products, SapCD, KO mice of SapC and SapCD were studied. While SapC seemed like a likely contributor to neuronal 5. Discussion: Saposins function in the overgrowth phenotype given the ear ‘neurotigenic’ portion of prosaposin is When one takes into account the found in the n-terminal region of SapC cumulative work on prosaposin and studies (O’Brien et al., 1994), this hypothesis was on saposins B, C and D, a number of not supported - SapC KO mice showed interesting generalizations emerge. normal hearing and morphology with no Ablation of prosaposin, as the precursor of obvious abnormalities in end-organs while all the saposins, causes the most significant SapCD KO mouse showed accumulation of defect in the inner ear (Akil et al., 2006; Akil inclusions in the vestibular system similar et al., 2012). This conforms to findings in but not severe to those seen in prosaposin other organs of mouse model of prosaposin KO mouse. ablation (Sun et al., 2002). Given the nature of the hearing loss, as well as efferent and While these vestibular findings, in isolation, afferent neuronal sprouting in the seem to identify lack of sapD as a likely prosaposin KO mouse, it was hypothesized cause of gait abnormalities, when compared that SapC, a protein known for its with systemic effects found in SapCD KO neurotigenic properties, was responsible mice, the abnormalities in the ear seem for these changes. However, the SapCD KO mild and thus it could not be concluded that mouse has minimal histologic changes, and loss of SapD alone was responsible. Sun et no effect on hearing. In contrast, though al. found a severe neurological phenotype in much milder than the full prosaposin null SapCD KO mice including ataxia, kyphotic mutation, the Sap B KO mouse has a more posturing, and hind limb paralysis. Further, severe deficit with hearing loss starting at storage bodies in the neurons of the spinal ~6 months and progressing thereafter. cord, brain, and dorsal root ganglion as well Thus SapB appears to have a much greater as accumulation of alpha-hydroxy role in auditory neuronal maintenance and ceramides and glucosylceramides in brain balance than SapC and/or D. and kidney were also seen. These central changes were more severe than those seen However, as others have noted, one cannot in the vestibular system in SapCD KO mice simply sum up the constituent saposins A-D and thus likely explain the gait to determine the overall role of prosaposin. abnormalities observed (Sun et al., 2007). Deficiencies in single saposin or combined In this model, beyond 6 weeks loss of saposins have exhibited significantly Purkinje cells was noted, along with storage different physiological effects than were bodies in neurons of the spinal cord, brain expected by summing the individual Copyright 2015 KEI Journals. All Rights Reserved Page | 10

Medical Research Archives deficiencies (Sun et al., 2007). Prosaposin findings in the prosaposin KO mice itself can act as neurite outgrowth or nerve phenotype is also not straightforward. As has regeneration factor or may be involved in been pointed out by Sun et al, in mice, preventing cell death (Hiraiwa et al., 1992; deficiencies of saposin A cause a Krabbe-like Kotani et al., 1996; Sun et al., 2002). It is demyelinating disease while loss of SapD thus possible that individual saposins may results in ceramide accumulation and bladder be acting synergistically with the defects; yet neither of these abnormalities are progenitor prosaposin to function effectively, and no ‘single’ saposin is responsible for maintenance of the auditory seen in the prosaposin KO mouse. Analogous system. discrepancies have been seen in SapB and C KO mice as compared to prosaposin KO mice Investigating a synergistic function as (Sun et al., 2002). Thus, deficiencies of opposed to individual roles is complicated by saposin A or D might similarly yield auditory the fact that prosaposin is also detected by and vestibular phenotypes markedly antibodies to individual saposins. This makes different than that seen in the prosaposin KO cellular localization of individual saposins not mouse. Table 1 summarized the auditory and currently possible, complicating the question vestibular findings in KO models of prosaposin, of which saposins are the most active in the SapA, SapB, SapC, SapD, SapC+D. inner ear. Additionally, assigning specific functions of individual saposins based on the Table 1. Summary of auditory and vestibular findings in KO models of prosaposin, SapA, SapB, SapC, SapD, SapC+D.

Knockout Auditory Findings Vestibular Findings

Model Prosaposin Important for normal cochlear Critical in neuronal maturation process of innervation and maintenance of normal vestibular sensory epithelium and Hearing maintenance of normal vestibular function

Expression of prosaposin observed in Prosaposin localized to three vestibular organ of Corti, supporting cells Deiters’ end-organs and Scarpa’s ganglion cells, IHCs, inner pillar cells, synaptic region of OHCs Vestibular dysfunction on behavioral tasks

Reduction in hearing in mice up to P19, Exuberant cellular proliferation below deafness by P25, reduced DPOAE from vestibular HCs with disruption of P15 onward supporting cells

Medical Research Archives

Cellular hypertrophy in IHCs and Inclusion bodies and cellular proliferation greater epithelial ridge, loss of OHCs disturbing normal neuroepithelial form apex, vacuolization of OHCs structure of vestibular end-organs corresponding with afferent and efferent Overgrowth of auditory afferent neuronal overgrowth neurites in region of IHCs and proliferation of auditory efferent neurites in region of tunnel of Corti

Normal HC function SapA Little is known. Not studied yet in the ear Little is known. Not studied yet in the vestibular SapB progressive sulfatide accumulation in Head tremor satellite cells around spiral ganglion (SG) neurons resulting in satellite cell No clear evidence for peripheral degeneration highly correlated to SG vestibular dysfunction phenotype degeneration, and ultimately, loss of Hearing Storage material accumulation in vestibular ganglion Normal organ of Corti morphology, significant reduction of SG and cochlear VsEPs showed profound vestibular nerve fibers in Rosenthal’s canals dysfunction likely from large-scale progress with age neuronal degeneration in myelinated SG neurons No neuronal overgrowth observed SapC No histologic changes in organ of Corti No effect on the vestibular function (No abnormal movement patterns, no inclusion No effect on hearing bodies) SapD Least understood. Not studied yet in the Suggested that lack of SapD is responsible Ear for vestibular abnormalities seen in SapCD KO mice Sap C+D No histologic changes in organ of Corti Difficulty walking, abnormal movement

No effect on hearing Inclusion bodies in SapCD KO mice in all three end-organs

Cellular proliferations consistent with neuronal overgrowth

Medical Research Archives

References 355.

Akil O, Chang J, Hiel H et al. Progressive D'Hooge R, Coenen R, Gieselmann V, deafness and altered cochlear innervation Lullmann-Rauch R, De Deyn PP. Decline in in knock-out mice lacking prosaposin. J brainstem auditory-evoked potentials Neuroscience 2006; 26: 13076-13088. coincides with loss of spiral ganglion cells in arylsulfatase A-deficient mice. Brain Res. Akil O, Lustig LR. Severe vestibular 1999; 847: 352-356. dysfunction and altered vestibular innervation in mice lacking prosaposin. Deconinck N, Messaaoui A, Ziereisen Fet Neuroscience research 2012; 72: 296-305. al. Metachromatic leukodystrophy without arylsulfatase A deficiency: a new case of Akil O, Sun Y, Zhang W, Ku T, Lee C, Jones S, saposin-B deficiency. Eur. J. Paediatr. Grabowski G, Lustig LR. Spiral Ganglion Neurol. 2008; 12: 46-50. Degeneration and Hearing Loss as a Consequence of Satellite Cell Death in de Hosson LD, van de Warrenburg BP, Saposin B-Deficient Mice. J Neuroscience Preijers FWet al. Adult metachromatic 2015; 35(7): 3263-3275. leukodystrophy treated by allo-SCT and a review of the literature. Bone Marrow Azuma N, O'Brien JS, Moser HW, Kishimoto Transplant 2011; 46: 1071-1076. Y. Stimulation of acid ceramidase activity by saposin D. Archives of biochemistry and Fabbro D, Grabowski GA. Human acid β- biophysics 1994; 311: 354-357. glucosidase: use of inhibitory and activating monoclonal antibodies to investigate the Bamiou DE, Campbell P, Liasis A et al. enzyme’s catalytic mechanism and saposin Audometric abnormalities in children with A and C binding sites. J. Biol. Chem 1991; Gaucher disease type 3. Neuropediatrics 266: 15021-15027. 2001; 32: 136-141. Fujita N, Suzuki K, Vanier MTet al. Targeted disruption of the mouse sphingolipid Campbell PE, Harris CM, Vellodi A. activator protein gene: a complex Deterioration of the auditory brainstem phenotype, including severe response in children with type 3 Gaucher leukodystrophy and wide-spread storage of disease. Neurology 2004; 63: 385-387. multiple sphingolipids. Human molecular genetics 1996; 5: 711-725. Ciaffoni F, Tatti M, Boe Aet al. Saposin B binds and transfers phospholipids. J Lipid Furst W, Sandhoff K. Activator proteins and Res. 2006; 47: 1045-1053 topology of lysosomal sphingolipid catabolism. Biochimica et biophysica acta Coenen R, Gieselmann V, Lullmann-Rauch 1992; 1126: 1-16. R. Morphological alterations in the inner ear of the arylsulfatase A-deficient mouse. Gao J, Yin D, Yao Y, Williams TD, Squier TC.

Acta neuropathologica 2001; 101: 491-498. Progressive decline in the ability of calmodulin isolated from aged brain to Colombaioni L, Garcia-Gil M. Sphngolipid activate the plasma mem-brane Ca-ATPase. metabolites in neural signaling and Biochemistry 1998; 37: 9536–9548 function. Brain Res. Rev. 2004; 46(3): 328- Copyright 2015 KEI Journals. All Rights Reserved Page | 13

Medical Research Archives

coding for a sphingolipid activator protein, Gieselmann V, Krageloh-Mann I. Meta SAP-1, is on human chromosome 10. chromatic leukodystrophy--an update. Human genetics 1985; 69: 197-200. Neuropediatrics 2010; 41: 1-6. Jatzkewitz H. Eine neue Methode zur Hakomori S. Chemistry of glycol quantitativen Ultramikrobestimmung der sphingolipids. Handbook of lipid research Sphingolipoide aus Gehirm. Hoppe-Seyler’s Z. 1983; 3: 1-164. Physiol. Chem. 1964; 336: 25-39.

Henseler M., Klein A., Glombitza G.J., Suzuki Kishimoto Y., Hiraiwa M., O’Brien J.S. K., Sandhoff K. Expression of the three Saposins: structure, function, distribution, alternative forms of the sphingolipid and molecular genetics. J. Lipid Res. 1992; activator protein precursor in baby 33: 1255–1267 hamster kidney cells and functional assays in a cell culture system. J. Biol. Chem. 1996; Kotani Y, Matsuda S, Sakanaka M, Kondoh 271: 8416–8423 K, Ueno S, Sano A. Prosaposin facilitates sciatic nerve regeneration in vivo. J Hess B, Saftig P, Hartmann Det al. Neurochem. 1996; 66: 2019-2025. Phenotype of arylsulfatase A-deficient mice: relationship to human metachromatic Kujawa SG, Liberman MC. Adding insult to leukodystrophy. Proceedings of the injury: cochlear nerve degeneration after National Academy of Sciences of the United "temporary" noise-induced hearing loss. J States of America 1996; 93: 14821-14826. Neurosci 2009; 29: 14077-14085.

Hineno, T., A. Sano, K. Kondoh, S. Ueno, Y. Land MF. Motion and vision: why animals Kakimoto, and K. Yoshida. Secretion of move their eyes. J Comp Physiol A. 1999; sphingolipid hydrolase activator precursor, 185: 341–352 prosaposin. Biochem. Biophys. Res. Commun. 1991; 176: 668-674. Li SC, Sonnino S, Tettamanti G, Li YT. Characterization of a nonspecific activator Hiraiwa M, Soeda S, Kishimoto Y, O'Brien protein for the enzymatic hydrolysis of JS. Binding and transport of gangliosides glycolipids. The Journal of biological by prosaposin. Proceedings of the National chemistry 1988; 263: 6588-6591. Academy of Sciences of the United States of America 1992; 89: 11254-11258. Lin HW, Furman AC, Kujawa SG, Liberman MC. Primary neural degeneration in the Holtschmidt H, Sandhoff K, Kwon HY, Guinea pig cochlea after reversible noise- Harzer K, Nakano T, Suzuki K. Sulfatide induced threshold shift. J Assoc. Res. activator protein. Alternative splicing that Otolaryngol. 2011; 12: 605-616. generates three mRNAs and a newly found mutation responsible for a clinical disease. The Journal of biological chemistry 1991; Lustig LR, Alemi S, Sun Y, Grabowski G, Akil O. Role of Saposin C and D in auditory and 266: 7556-7560. vestibular function. Laryngoscope. 2015 Jul 21. doi: 10.1002/lary.25479. Inui K, Kao FT, Fujibayashi Set al. The gene Copyright 2015 KEI Journals. All Rights Reserved Page | 14

Medical Research Archives

Marzella PL, Clark GM. Growth factors, O'Brien JS, Carson GS, Seo HC, Hiraiwa M, auditory neurones and cochlear implants: a Kishimoto Y. Identification of prosaposin as review. Acta Otolaryngol. 1999; 119: 407– a neurotrophic factor. Proceedings of the 412. National Academy of Sciences of the United States of America 1994; 91: 9593-9596. Matsuda J, Kido M, Tadano-Aritomi Ket al. Mutation in saposin D domain of O'Brien JS, Carson GS, Seo HCet al. sphingolipid activator protein gene causes Identification of the neurotrophic factor urinary system defects and cerebellar sequence of prosaposin. Faseb. J. 1995; 9: Purkinje cell degeneration with 681-685. accumulation of hydroxy fatty acid- containing ceramide in mouse. Human Paton BC, Schmid B, Kustermann-Kuhn B, molecular genetics 2004; 13: 2709-2723. Poulos A, Harzer K. Additional biochemical findings in a patient and fetal sibling with a Morimoto S, Martin BM, Kishimoto Y, genetic defect in the sphingolipid activator O'Brien JS. Saposin D: a sphingomyelinase protein (SAP) precursor, prosaposin. activator. Biochemical and biophysical Evidence for a deficiency in SAP-1 and for a research communications 1988; 156: 403- normal lysosomal neuraminidase. Biochem 410. J 1992; 285 (2): 481-488.

Morimoto S, Martin BM, Yamamoto Y, Kretz Radin, N. S. The cohydrolases for KA, O'Brien JS, Kishimoto Y. Saposin A: cerebroside 0-glucosidase. In The second cerebrosidase activator protein. Molecular Basis of Lysosomal Storage Proceedings of the National Academy of Disorders. R. 0. Brady and J. A. Barranger, Sciences of the United States of America editors. Academic Press, New York. 1984; 1989; 86: 3389-3393. 93-112.

Nakaizumi T, Kawamoto K, Minoda R, Sandhoff K. The hydrolysis of Tay-Sachs Raphael Y. Adenovirusmediated ganglioside (TSG) by human N-acetyl-β- expression of brain-derived neurotrophic D-hexosaminidase A. FEBS Lett. 1970; 11: factor protects spiral ganglion neurons 342–344 from ototoxic damage. Audiol. Neurootol.

2004; 9: 135–143. Sandhoff K, van Echten G, Schroder M, Schnabel D, Suzuki K. Metabolism of O'Brien JS, Kretz KA, Dewji N, Wenger DA, glycolipids: the role of glycolipid-binding Esch F, Fluharty AL. Coding of two proteins in the function and sphingolipid activator proteins (SAP-1 and pathobiochemistry of lysosomes. Biochem. SAP-2) by same genetic locus. Science 1988; Soc. Trans. 1992; 20: 695-699. 241: 1098-1101. Sandhoff K, Kolter T, Van Echten-Deckert O'Brien JS, Kishimoto Y. Saposin proteins: G. Sphingolipid metabolism. Sphingoid structure, function, and role in human analogs, sphingolipid activator proteins, lysosomal storage disorders. FASEB J 1991; and the pathology of the cell. Annals of the 5: 301-308. New York Academy of Sciences 1998;

Medical Research Archives

845: 139-151. Sun Y, Witte DP, Jin P, Grabowski GA. Sandhoff K. The GM2-gangliosidoses and Analyses of temporal regulatory elements the elucidation of the beta-hexosaminidase of the prosaposin gene in transgenic mice. system. Adv Genet 2001; 44: 67-91. Biochem J 2003; 370: 557-566.

Sandhoff K. My journey into the world of Sun Y, Witte DP, Zamzow Met al. Combined sphingolipids and sphingolipidoses. Proc saposin C and D deficiencies in mice lead to Jpn Acad Ser B Phys Boil Sci. 2012; 88(10): a neuronopathic phenotype, 554-82. glucosylceramide and alpha-hydroxy ceramide accumulation, and altered Schlote W., Harzer K., Christomanou H., prosaposin trafficking. Human molecular Paton B.C., Kustermann-Kuhn B., Schmid B., genetics 2007; 16: 957-971. Seeger J., Beudt U., Schuster I., Langenbeck U. Sphingolipid activator protein 1 Sun Y, Witte DP, Ran Het al. Neurological deficiency in metachromatic deficits and glycosphingolipid accumulation leucodystrophy with normal arylsulphatase in saposin B deficient mice. Human A activity. A clinical, morphological, molecular genetics 2008; 17: 2345-2356. biochemical and immunological study. Eur. J. Pediatr. 1991; 150: 584–591 Terashita T, Saito S, Miyawaki Ket al. Localization of prosaposin in rat cochlea.

Neuroscience research 2007; 57: 372-378 Shepherd RK, Hardie NA Deafness-induced changes in the auditory pathway: Wenger DA, DeGala G, Williams Cet al. implications for cochlear implants. Audiol Neurootol. 2001; 6: 305–318 Clinical, pathological, and biochemical studies on an infantile case of Spoor F, Bajpai S, Hussain ST, Kumar K, sulfatide/GM1 activator protein deficiency. Thewissen JG. Vestibular evidence for the Am J Med Genet 1989; 33: 255-265. evolution of aquatic behaviour in early cetaceans. Nature. 2002; 417: 163–166 Walls G. The evolutionary history of eye movements. Vision Research. 1962; 2: 69– Sundaram SK, Fan JH, Lev M. A neutral 80. galactocerebroside sulfate sulfatidase from mouse brain. J Biol Chem 1995; 270: 10187-10192 Zhang XL, Rafi MA, DeGala G, Wenger DA. Insertion in the mRNA of a metachromatic Sun Y, Qi X, Witte DPet al. Prosaposin: leukodystrophy patient with sphingolipid threshold rescue and analysis of the activator protein-1 deficiency. Proceedings "neuritogenic" region in transgenic mice. of the National Academy of Sciences of the Molecular genetics and metabolism 2002; United States of America 1990; 87: 1426- 76: 271-286. 1430.