Hiroaki Nabeka

Attenuation of MPTP/MPP+ toxicity in vivo and in vitro by an 18-mer peptide derived from prosaposin

Parkinsons disease (PD) is a chronic progressive neurological disorder with an increasing incidence in the aging population. Neuroprotective and/or neuroregenerative strategies remain critical in the treatment of this increasingly prevalent disease. Prosaposin is a neurotrophic factor whose neurotrophic activity is attributed to a stretch of 12 amino acids located at the N-terminal region of saposin C. The present study was performed to investigate the protective effect and mechanism of action of a prosaposin-derived 18-mer peptide (PS18: LSELIINNATEELLIKGL) in Parkinsons disease models. We used 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or 1-methyl-4-phenylpyridinium ion (MPP(+))-induced dopaminergic neurotoxicity in C57BL/6J mice or SH-SY5Y cells and explored the protective effect and mechanisms of action of PS18 on dopaminergic neurons. Treatment with 2.0mg/kg PS18 significantly improved behavioral deficits, enhanced the survival of tyrosine hydroxylase-positive neurons, and decreased the activity of astrocytes in the substantia nigra and striatum in MPTP-induced PD model mice. In vitro, a Cell Counting Kit-8 assay and Hoechst 33258 staining revealed that co-treatment with 300ng/mL PS18 and 5mM MPP(+) protected against MPP(+)-induced nuclear morphological changes and attenuated cell death induced by MPP(+). We also found that PS18-FAM entered the cells, and the retention time of PS18-FAM in the cytoplasm of MPP(+)-treated cells was shorter than that of untreated cells. In addition, PS18 showed protection from MPP(+)/MPTP-induced apoptosis in the SH-SY5Y cells and dopaminergic neurons in the PD model mice via suppression of the c-Jun N-terminal kinase/c-Jun pathway; upregulation of Bcl-2; downregulation of BAX, attenuating mitochondrial damage; and inhibition of caspase-3. These findings suggest that PS18 may provide a valuable therapeutic strategy for the treatment of progressive neurodegenerative diseases such as PD.

Rho kinase inhibitors stimulate the migration of human cultured osteoblastic cells by regulating actomyosin activity.

We investigated the effects of Rho-associated kinase (ROCK) on migration and cytoskeletal organization in primary human osteoblasts and Saos-2 human osteosarcoma cells. Both cell types were exposed to two different ROCK inhibitors, Y-27632 and HA-1077. In the improved motility assay used in the present study, Y-27632 and HA-1077 significantly increased the migration of both osteoblasts and osteosarcoma cells on plastic in a dose-dependent and reversible manner. Fluorescent images showed that cells of both types cultured with Y-27632 or HA-1077 exhibited a stellate appearance, with poor assembly of stress fibers and focal contacts. Western blotting showed that ROCK inhibitors reduced myosin light chain (MLC) phosphorylation within 5 min without affecting overall myosin light-chain protein levels. Inhibition of ROCK activity is thought to enhance the migration of human osteoblasts through reorganization of the actin cytoskeleton and regulation of myosin activity. ROCK inhibitors may be potentially useful as anabolic agents to enhance the biocompatibility of bone and joint prostheses.

Prosaposin expression in the regenerated muscles of mdx and cardiotoxin-treated mice.

The trophic factor prosaposin (PS) is strongly expressed in skeletal muscle, and reportedly, a PS-derived peptide attenuates loss of muscle mass after nerve injury in vivo and increases myoblast fusion into myotubes in vitro. However, few studies have focused on the role of PS during muscle regeneration. We examined the expression of PS in the skeletal muscles in normal, mdx, and cardiotoxin (CTX)-treated mice using immunofluorescence staining, Western blotting, and in situ hybridisation. Immunofluorescence showed intense PS immunoreactivity in the peripheral cytoplasm of uninjured myofibres of normal mice and regenerated myofibres of 8 weeks post-CTX-injection mice. In early stage CTX-treated mice (14 days and earlier), intense PS immunoreactivity was also detected in the immune cells that infiltrated damaged muscle, but it was weak for regenerating myofibres. Western blot confirmed these findings. In contrast, PS was continuously low in mdx mice in both immunofluorescence and Western blotting. In situ hybridisation confirmed the decrease of PS mRNA in regenerated myofibres and revealed the main form of PS mRNA as Pro+0 without a 9-base insertion both in normal and mdx mice. The embryonic myosin (MYH3) was clearly localized in the newly regenerated myofibres at 3, 7, and 14 days of post-CTX-injection and mdx mice, but was lower in the late stage of regenerated myofibres (28 and 56 days post-CTX injection). The inverse distribution of MYH3 and PS indicates that the PS expression is closely related to the differentiation of regenerated myofibres. Investigation of the mitogen-activated protein (MAP) kinase signal pathway showed the inversely synchronous correlation of phosphorylated ERK1/2 with myofibre PS and the synchronous correlation of phosphorylated p-38 with myofibre PS. These data suggest that PS is involved in the regulation of muscle differentiation of regenerated fibres.

Decrease in Prosaposin in the Dystrophic mdx Mouse Brain

Background Duchenne muscular dystrophy caused by a mutation in the X-linked dystrophin gene induces metabolic and structural disorders in the brain. A lack of dystrophin in brain structures is involved in impaired cognitive function. Prosaposin (PS), a neurotrophic factor, is abundant in the choroid plexus and various brain regions. We investigated whether PS serves as a link between dystrophin loss and gross and/or ultrastructural brain abnormalities. Methodology/Principal Findings The distribution of PS in the brains of juvenile and adult mdx mice was investigated by immunochemistry, Western blotting, and in situ hybridization. Immunochemistry revealed lower levels of PS in the cytoplasm of neurons of the cerebral cortex, hippocampus, cerebellum, and choroid plexus in mdx mice. Western blotting confirmed that PS levels were lower in these brain regions in both juveniles and adults. Even with low PS production in the choroids plexus, there was no significant PS decrease in cerebrospinal fluid (CSF). In situ hybridization revealed that the primary form of PS mRNA in both normal and mdx mice was Pro+9, a secretory-type PS, and the hybridization signals for Pro+9 in the above-mentioned brain regions were weaker in mdx mice than in normal mice. We also investigated mitogen-activated protein kinase signalling. Stronger activation of ERK1/2 was observed in mdx mice, ERK1/2 activity was positively correlated with PS activity, and exogenous PS18 stimulated both p-ERK1/2 and PS in SH-SY5Y cells. Conclusions/Significance Low levels of PS and its receptors suggest the participation of PS in some pathological changes in the brains of mdx mice.

Distribution of prosaposin in rat lymphatic tissues

Prosaposin (PSAP) is as a trophic factor and an activator protein for sphingolipid hydrolase in lysosomes. We generated a specific antibody to PSAP and examined the spatiotemporal distribution of PSAP-immunoreactive (PSAP-IR) cells in the lymphatic tissues of Wistar rats. Immunoblots of tissue homogenates separated electrophoretically showed a single band for PSAP in brain but two bands in spleen. PSAP-IR cells were distributed in both the red and white pulp of the spleen, in both the cortex and medulla of the thymus and in mesenteric lymph nodes. Many PSAP-IR cells were found in the dome portion of Peyer’s patches and the number of PSAP-IR cells increased with the age of the rat. To identify the PSAP-IR cells, double- and triple-immunostainings were performed with antibodies against PSAP, CD68 and CD1d. The large number of double- and triple-positive cells suggested that antigen-presenting cells contained much PSAP in these lymphatic tissues. Intense expression of PSAP mRNA, examined by in situ hybridisation, was observed in the red pulp and corona of the spleen. In rats, the PSAP gene generates two alternative splicing forms of mRNA: Pro+9 containing a 9-base insertion and Pro+0 without the insertion. We examined the expression patterns of the alternative splicing forms of PSAP mRNA in the spleen. The presence of both types of mRNA (Pro+9 and Pro+0) indicated that the spleen contains various types of prosaposin-producing and/or secreting cells. These findings suggest diverse functions for PSAP in the immune system.

Prosaposin Overexpression following Kainic Acid-Induced Neurotoxicity

Because excessive glutamate release is believed to play a pivotal role in numerous neuropathological disorders, such as ischemia or seizure, we aimed to investigate whether intrinsic prosaposin (PS), a neuroprotective factor when supplied exogenously in vivo or in vitro, is up-regulated after the excitotoxicity induced by kainic acid (KA), a glutamate analog. In the present study, PS immunoreactivity and its mRNA expression in the hippocampal and cortical neurons showed significant increases on day 3 after KA injection, and high PS levels were maintained even after 3 weeks. The increase in PS, but not saposins, detected by immunoblot analysis suggests that the increase in PS-like immunoreactivity after KA injection was not due to an increase in saposins as lysosomal enzymes after neuronal damage, but rather to an increase in PS as a neurotrophic factor to improve neuronal survival. Furthermore, several neurons with slender nuclei inside/outside of the pyramidal layer showed more intense PS mRNA expression than other pyramidal neurons. Based on the results from double immunostaining using anti-PS and anti-GABA antibodies, these neurons were shown to be GABAergic interneurons in the extra- and intra-pyramidal layers. In the cerebral cortex, several large neurons in the V layer showed very intense PS mRNA expression 3 days after KA injection. The choroid plexus showed intense PS mRNA expression even in the normal rat, and the intensity increased significantly after KA injection. The present study indicates that inhibitory interneurons as well as stimulated hippocampal pyramidal and cortical neurons synthesize PS for neuronal survival, and the choroid plexus is highly activated to synthesize PS, which may prevent neurons from excitotoxic neuronal damage. To the best of our knowledge, this is the first study that demonstrates axonal transport and increased production of neurotrophic factor PS after KA injection.

Chronological changes in prosaposin in the developing rat brain.

Prosaposin is the precursor protein of four glycoproteins, saposins A, B, C, and D, which activate sphingolipid hydrolases in lysosomes. Besides this role, intact prosaposin is also known as a potent neurotrophic factor that prevents neuronal cell death and stimulates neurite outgrowth in in vivo and in vitro experiments. In the present study, we examined chronological changes in prosaposin immunoreactivity in the rat brain using immunofluorescence staining and Diaminobenzidine (DAB) immunohistochemistry. In the hippocampal regions CA1, CA3, and dentate gyrus, the strongest staining of prosaposin was observed on postnatal day 1. The prosaposin immunoreactivity then decreased gradually until postnatal day 28. But in the cerebral cortex, prosaposin staining intensity increased from postnatal day 1 to 14, then decreased until postnatal day 28. The prosaposin immunoreactivity co-localized with the lysosomal granules labeled by an anti-Cathepsin D antibody, indicating that prosaposin mainly localized in the lysosomes of the neurons. We also examined the chronological changes in prosaposin mRNA and its two alternatively spliced variants using in situ hybridization. We found that both the mRNA forms, especially the one without a nine-base insertion, increased significantly from embryonic day 15 to postnatal day 7, then decreased gradually until postnatal day 28. Abundant prosaposin expression in the perinatal stages indicates a potential role of prosaposin in the early development of the rat brain.

A prosaposin-derived Peptide alleviates kainic Acid-induced brain injury.

Four sphingolipid activator proteins (i.e., saposins A–D) are synthesized from a single precursor protein, prosaposin (PS), which exerts exogenous neurotrophic effects in vivo and in vitro. Kainic acid (KA) injection in rodents is a good model in which to study neurotrophic factor elevation; PS and its mRNA are increased in neurons and the choroid plexus in this animal model. An 18-mer peptide (LSELIINNATEELLIKGL; PS18) derived from the PS neurotrophic region prevents neuronal damage after ischemia, and PS18 is a potent candidate molecule for use in alleviating ischemia-induced learning disabilities and neuronal loss. KA is a glutamate analog that stimulates excitatory neurotransmitter release and induces ischemia-like neuronal degeneration; it has been used to define mechanisms involved in neurodegeneration and neuroprotection. In the present study, we demonstrate that a subcutaneous injection of 0.2 and 2.0 mg/kg PS18 significantly improved behavioral deficits of Wistar rats (n = 6 per group), and enhanced the survival of hippocampal and cortical neurons against neurotoxicity induced by 12 mg/kg KA compared with control animals. PS18 significantly protected hippocampal synapses against KA-induced destruction. To evaluate the extent of PS18- and KA-induced effects in these hippocampal regions, we performed histological evaluations using semithin sections stained with toluidine blue, as well as ordinal sections stained with hematoxylin and eosin. We revealed a distinctive feature of KA-induced brain injury, which reportedly mimics ischemia, but affects a much wider area than ischemia-induced injury: KA induced neuronal degeneration not only in the CA1 region, where neurons degenerate following ischemia, but also in the CA2, CA3, and CA4 hippocampal regions.

Differential expression of the alternatively spliced forms of prosaposin mRNAs in rat choroid plexus

Prosaposin has two distinct profiles. One is a precursor form that is processed into saposins thus promoting lysosomal sphingolipid hydrolase function, whereas the other is an intact form that is not processed into saposins but is abundant in certain tissues and secretory fluids, including the cerebrospinal fluid. In rats, alternative splicing in the prosaposin gene generates mRNAs with and without a 9-base insertion (Pro+9 and Pro+0 mRNAs, respectively). Pro+9 mRNA is reported to be preferentially expressed in tissues in which the intact form of prosaposin dominates, whereas Pro+0 mRNA is preferentially expressed in tissues in which the precursor dominates. The expression patterns of Pro+9 and Pro+0 mRNAs in the rat choroid plexus are examined in the present study. The specificities of 36-mer oligonucleotide probes used to detect the 9-base insertion by in situ hybridization were demonstrated by dot-blot hybridization. Next, these probes were used for in situ hybridization, which showed predominant expression of Pro+0 mRNA and weak expression of Pro+9 mRNA in the choroid plexus. These expression patterns were confirmed by reverse transcription plus the polymerase chain reaction with AlwI restriction enzyme treatment. Expression of the intact form of prosaposin in the choroid plexus was assessed by Western blotting and immunohistochemistry. Because the choroid plexus is responsible for the generation of cerebrospinal fluid containing the intact form of prosaposin, the present study raises the possibility that Pro+0 mRNA is related to the intact form in the choroid plexus and that the alternatively spliced forms of mRNAs do not simply correspond to the precursor and intact forms of prosaposin.

Prosaposin-derived peptide alleviates ischaemia-induced hearing loss.

Abstract Conclusion: An 18-mer peptide derived from the neurotrophic region of prosaposin (PS-pep) prevents hearing loss and cochlear damage due to transient cochlear ischaemia by activating an anti-apoptotic pathway. PS-pep is a potent candidate molecule for alleviating ischaemia-induced hearing loss. Objective: PS-pep was investigated for its protective effects against ischaemia-induced hearing loss and cochlear damage. Methods: Ischaemia was induced in both cochleae in Mongolian gerbils by pulling the ligatures around both vertebral arteries in an anterior direction using 5 g weights for 15 min. PS-pep was synthesized artificially and administered subcutaneously four times after the induction of transient cochlear ischaemia. Results: An increase in the auditory brainstem response threshold was alleviated in animals treated with 2.0 mg/kg PS-pep. Histological examinations conducted on day 7 showed that the loss of inner hair cells (IHCs) was more prominent than that of outer hair cells. Higher doses of PS-pep significantly alleviated IHC loss. An increase in the anti-apoptotic factor bcl-2 was also noted in the IHCs treated with PS-pep.