JOURNAL OF NEUROCHEMISTRY

| 2014 | 131 | 101–114

doi: 10.1111/jnc.12782

, ,

*Department of Neurology, University of Mississippi Medical Center, Jackson, Mississippi, USA †Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi, USA

Abstract Spinocerebellar ataxia-1 (SCA1) is a neurodegenerative disease that primarily targets Purkinje cells (PCs) of the cerebellum. The exact mechanism of PC degeneration is unknown, however, it is widely believed that mutant ataxin-1 becomes toxic because of the phosphorylation of its serine 776 (S776) residue by cAMP-dependent protein kinase A (PKA). Therefore, to directly modulate mutant ATXN1 S776 phosphorylation and aggregation, we designed a therapeutic polypeptide to inhibit PKA. This polypeptide comprised of a thermally responsive elastin-like peptide (ELP) carrier, which increases peptide half-life, a PKA inhibitory peptide (PKI), and a cell-penetrating peptide (Synb1). We observed that our therapeutic polypeptide, Synb1-ELP-PKI, inhibited PKA activity at concentrations similar to the PKI peptide. Additionally,

Synb1-ELP-PKI significantly suppressed mutant ATXN1 S776 phosphorylation and intranuclear inclusion formation in cell culture. Further, Synb1-ELP-PKI treatment improved SCA1 PC morphology in cerebellar slice cultures. Furthermore, the Synb1-ELP peptide carrier crossed the blood–brain barrier and localized to the cerebellum via the i.p. or intranasal route. Here, we show the intranasal delivery of ELP-based peptides to the brain as a novel delivery strategy. We also demonstrate that our therapeutic polypeptide has a great potential to target the neurotoxic S776 phosphorylation pathway in the SCA1 disease. Keywords: ataxin-1 phosphorylation, intranasal peptide delivery, neurodegeneration, PKA inhibitory peptide, Purkinje cell, spinocerebellar ataxia-1. J. Neurochem. (2014) 131, 101–114.

Spinocerebellar ataxia-1 (SCA1) is a neurological disorder, resulting from a CAG repeat expansion in the ataxin-1 (ATXN1) gene (Koeppen 2005; Orr and Zoghbi 2007; Matilla-Duenas et al. 2008). The polyglutamine expanded mutant ATXN1 primarily targets Purkinje cells (PCs) of the cerebellum (Koeppen 2005; Orr and Zoghbi 2007; MatillaDuenas et al. 2008). The most widely accepted mechanism of PC degeneration in SCA1 is centered around the phosphorylation of the serine 776 (S776) residue on the mutant ATXN1 protein (Chen et al. 2003; Emamian et al. 2003; Jorgensen et al. 2009; Hearst et al. 2010). The mutant ATXN1 S776 residue is phosphorylated by cAMPdependent protein kinase A (PKA) and stabilized by binding of the 14-3-3 protein to resist protein degradation (Chen et al. 2003; Jorgensen et al. 2009; Hearst et al. 2010). The S776 phosphorylation of mutant ATXN1 makes nuclear inclusions both in cell culture and mouse models, which become toxic to cells (Chen et al. 2003; Jorgensen et al. 2009; Hearst et al. 2010). The expression of the mutant ATXN1 protein in SCA1 transgenic (Tg) mice also

causes the formation of cytoplasmic PC vacuoles containing the Bergmann glial protein S100B (Skinner et al. 2001; Vig et al. 2006a, 2009). These vacuoles contain degrading as well as membrane proteins such as protein kinase Cc, glutamate receptors, myo-inositol monophosphatase 1, and dopamine 2 receptors (D2R) (Hearst et al. 2010). D2Rs are G-protein-coupling receptors that inhibit adenylate cyclase and cAMP accumulation to regulate PKA activity (Neve et al. 2004; Liu et al. 2008). Previously, we proposed that

Received December 5, 2013; revised manuscript received May 13, 2014; accepted June 2, 2014. Address correspondence and reprint requests to Parminder J. S. Vig, Department of Neurology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216, USA. E-mail: pvig@ umc.edu Abbreviations used: ATXN1, ataxin-1; CaB, calbindin; D2R, dopamine receptor D2; EC50, half maximal effective concentration; ELP, elastin-like polypeptide; FSK, forskolin; GFP, green fluorescent protein; IN, intranasal; PC, Purkinje cell; PKA, cAMP-dependent protein kinase; Rho, rhodamine; S776, serine 776; SCA1, Spinocerebellar ataxia-1.

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 131, 101--114

101

102

S. M. Hearst et al.

vacuolar formation leads to the loss of the D2R altering PKA regulation in SCA1 PCs, causing an increase in ATXN1 S776 phosphorylation and aggregation (Hearst et al. 2010). The objective of this study was to design a therapeutic peptide to target PKA in SCA1 PCs and directly influence ATXN1 S776 phosphorylation and aggregation. Our therapeutic polypeptide was designed using three key elements: (i) the elastin-like polypeptide (ELP), a thermally responsive polypeptide, (ii) the PKA inhibitory peptide (PKI), a known PKA inhibitory peptide, (iii) a cellpenetrating peptide, Synb1, to enhance intracellular delivery. The ELP polypeptide is derived from a sequence of mammalian elastin (Ong et al. 2006). There are several advantages of using ELP-based polypeptides as therapeutic peptide carrier. ELPs consist of Val-Pro-Gly-Xaa-Gly repeated units, and undergo an inverse temperature transition (McPherson et al. 1992; Urry 1992). Below a characteristic transition temperature, ELPs are structurally disordered and highly soluble. When the temperature is raised above their transition temperature, they undergo a sharp phase transition, leading to the aggregation of the biopolymer (McPherson et al. 1992; Urry 1992). This process is fully reversible, which allows for easy and high-purity peptide purification by only using simple heating and cooling methods (McPherson et al. 1992; Urry 1992). Also, ELP polymers can be expressed in E. coli, and large quantities of the molecule may be purified by simple thermal cycling (Bidwell and Raucher 2005). ELPs are genetically encoded, providing control over the ELP sequence and molecular weight to an extent that is impossible with synthetic polymer analogs (Bidwell and Raucher 2005). The macromolecular chain length and polydispersity control the residence time of the drug–polymer conjugate in systemic circulation (Allen 1998; Kissel et al. 2001). Previous studies have demonstrated that ELP increases peptide half-life, making the peptide more resistant to proteolysis, which are desirable characteristics in a peptide delivery agent (Liu et al. 2006; Bidwell and Raucher 2010; Bidwell et al. 2012). The Synb1 peptide was placed on the N-terminus of our therapeutic peptide, which is derived from naturally occurring protegrin peptides and mediates the delivery of peptides or compounds across the blood–brain barrier (Rousselle et al. 2000; Sarantseva et al. 2009). On the C-terminus, our therapeutic peptide is fused to a PKA inhibitory peptide, PKI. The PKI peptide was derived from the PKIa protein that interacts with the catalytic subunit of PKA inhibiting kinase activity (Demaille et al. 1977; Glass et al. 1992). PKIa is highly expressed in both skeletal and cardiac muscle as well as the cortex and the cerebellum (Van Patten et al. 1992). In this study, we have designed an ATXN1 S776 phosphorylation inhibitory polypeptide and demonstrated its therapeutic potential in vitro. Furthermore, we have established that our peptide carrier has a great potential to target therapeutics in vivo to the cerebellum,

which could be used to combat neurodegenerative pathways in SCA1 or other cerebellar ataxias.

Methods Materials Green fluorescent protein (GFP) antibody was purchased from Roche (Indianapolis, IN, USA). The S100B antibody was purchased from Abcam (Cambridge, MA, USA). Calbindin D-28K (CaB) antibody was obtained from Millipore (Temecula, CA, USA). Fetal bovine serum was purchased from HyClone (Logan, UT, USA). Dulbecco’s modified Eagle’s medium media and other chemical reagents were purchased from Fisher (Houston, TX, USA). Polypeptide production ELP was modified by the addition of the Synb1 peptide (RGGRLSYSRRRFSTSTGR) and PKI peptide (TYADFIASGRT GRRNAI) to produce Synb1-ELP-PKI by molecular cloning in a pET25b+ expression vector by recursive directional ligation and selected by ampicillin resistance as previously described (Meyer and Chilkoti 1999; Hearst et al. 2011). Constructs were verified by DNA sequencing. The ELP polypeptide is commonly used as a control peptide (Massodi et al. 2009; Moktan et al. 2012), where the polypeptides are labeled on the C terminal cysteine residue with a fluorescent dye (rhodamine or Alexa 750, Abcam, Cambridge, MA, USA) to monitor the impact of the cell-penetrating peptide on fluorescent ELP localization in vivo and in vitro (Massodi et al. 2009; Moktan et al. 2012; Hearst et al. 2011; Bidwell et al. 2013). ELP constructs were expressed using the pET25 expression system and purified by thermal cycling as described previously (Meyer and Chilkoti 1999; Bidwell and Raucher 2005; Hearst et al. 2011). The integrity of the purified peptides was checked by a sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel stained using Comassie blue for the Synb1-ELP and Synb1-ELP-PKI polypeptides. PKA activity assay The PKA inhibitory peptide PKI was purchased from Sigma, St Louis, MO, USA and diluted according to the manufacturer and stored at 80°C. The PKA activity assay kit was purchased from Enzo Life Sciences, Farmingdale, NY, USA. This kit contains a 96-well plate covered in a PKA substrate, where the level of phosphorylated substrate is detected using a phospho-specific antibody that causes a colorimetric absorbance change at 450 nm. The absorbance change at 450 nm is equivalent to the PKA activity in the given sample. 400 Units of active PKA was purchased from Sigma and diluted with 1 ml of kinase buffer provided in the PKA activity assay kit. 4.0 Units of active PKA was pre-incubated with 0.5 lM to 20.0 lM (0.5, 1.0, 2.5, 5.0, 10.0, and 20.0 lM) PKI peptide alone, Synb1-ELP-PKI or 10 lM Synb1-ELP for 15 min at 25°C. Samples with no inhibitors added were used as the control. The samples were then added to the 96-well plate and the standard PKA activity assay protocol was followed according to the manufacturer’s instructions. PKA activity was measured by the absorbance change at 450 nm. The absorbance values were then normalized to the control, where PKA activity for the control was equal to 4.0 Units. These data was plotted in Excel, where EC50 values were estimated by using the titration curve.

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 131, 101--114

PKA inhibitory polypeptide to treat SCA1

Cell culture studies Stable GFP-ATXN1[82Q] HEK cells were placed on CC2 chamber slides (Fisher) and serum starved overnight to reduce inclusion formation as described previously (Hearst et al. 2010; Vig et al. 2011). Cells were treated with serum-free media as Control, or pretreated for 2 h with 50 lM Synb1-ELP, the control peptide or 50 lM Synb1-ELP-PKI. Next, the cells were washed with phosphate-buffered saline and treated with 60 lM forskolin (FSK) or serum-free media for 4 h and then fixed with 4% paraformaldehyde for fluorescent microscopic analysis. Cells were visualized under an Olympus BX60 epifluorescence microscope (Olympus, Center Valley, PA, USA). GFP-ATXN1[82Q] expressing cells have been reported to display three phenotypes: soluble diffuse localization within the nucleus, small punctate foci or larger inclusions (Parfitt et al. 2009; Hearst et al. 2010). GFP-ATXN1[82Q] HEK cells under the various treatment conditions were scored as soluble diffuse localization within the nucleus, small punctate foci or larger inclusions, and the cell percentage taken as described previously (Hearst et al. 2010; Vig et al. 2011). Experiments were repeated at least three times and for statistical analysis, at least 100 cells per experiment (n = 100) were counted. Graphs were produced in Excel and show the Mean  SE. Stable GFP-ATXN1[82Q] HEK cells were placed in 6-well plates and treated as described in the above experiments, also the cells were pre-treated with 10 lM H89 for 2 h followed by 60 lM FSK for 4 h; H89 is a known PKA inhibitor previously reported to reduce ATNX1 S776 phosphorylation (Hearst et al. 2010). After treatment completion, the cells were lysed for western blot analysis. About 5.0 lgs of protein was loaded in each well of 4–20% acrylamide gel (Bio-Rad Laboratories, Hercules, CA, USA). The blot was probed with ATXN1 Phospho S776 antibodies (Abcam) to detect the level of ATNX1 S776 phosphorylation and then reprobed with GFP antibodies to detect the total level of GFP-ATXN1[82Q] protein. The optical densities of the protein bands were measured using Image J software (NIH freeware, National Institutes of Health, Bethesda, MA, USA). Graphs were generated in Excel and show the optical density ratio of Phospho S776/GFP normalized to the Control, where Controls was given a value of 1.0. Cerebellar slice cultures Wild-type (WT, FVB) mice were obtained from Jackson Labs, Bar Harbor, ME, USA. GFP-WT and GFP-SCA1 mice are grown in our animal facility as previously described (Vig et al. 2011). All animal protocols were approved by Institutional Animal Care and Use Committee at the University of Mississippi Medical Center. The GFP is expressed only in PCs of GFP-WT and GFP-SCA1 mice; transgene expression of the enhanced GFP gene is under the control of pcp2/L7 promoter similar to the ATXN1 expression in SCA1 mice (Burright et al. 1995; Vig et al. 2011). Cerebellar PC cultures were prepared from WT, GFP-WT or GFP-SCA1 Tg mice, where whole cerebellums were removed from 9-day-old mouse pups as previously described (Hearst et al. 2010; Vig et al. 2011). Meninges were carefully removed using a dissection microscope. Tissue was rinsed and place in Petri dishes containing 5% glucose and cold Gey’s balanced salt solution (Gey solution, Sigma). Cerebellar tissues were cut into 300 lm slice sections using a McIllwain tissue chopper (Stoelting Co., Wood Dale, IL, USA). Cerebellar slices were suspended in cold Gey’s solution containing 5% glucose, then

103

grown on Millicell membrane inserts (Fisher) using 6-well culture plate containing 1 ml plating media (vol/vol: 5% 10X Basal Medium with Earle’s Salt, 2.5% 10X Hank’s balanced salt solution, 25% Horse Serum (Invitrogen, Carlsbad, CA, USA), 1% 100X PenStrep-Glutamine, 4.5% 10% D-Glucose, 0.5% 7.5% Sodium bicarbonate and 61.5% sterile water, Sigma). Tissue cultured plates were incubated overnight at 35.5°C with 5% CO2 and 100% humidity. The next day, cells were washed and treated with various concentrations of peptides in the plating media. WT type slices were treated with 10.0 lM rhodamine labeled Synb1-ELP for 4, 8, 16 and 24 h. GFP-WT slices were used to test the toxicity of our therapeutic peptides at a high dose. GFP-WT slices were treated with media as Control, 10.0 lM Synb1-ELP, Synb1-ELP-TRTK or Synb1-ELP-PKI every 48 h for 10 days. GFP-SCA1 slices were treated with media as Control or 2.5 lM and 5.0 lM Synb1-ELP, Synb1-ELP-TRTK or Synb1-ELP-PKI every 48 h for 6 days. Slice cultures were fixed in 4% paraformaldehyde, then immuostained using CaB, S100B or GFP antibodies followed by fluorescent secondary antibodies (Sigma). PCs were visualized under an Olympus BX60 epifluorescence microscope. Digital images were analyzed with ImageJ software to measure changes in PC dendritic growth area as described previously (Hearst et al. 2010; Vig et al. 2011). Tertiary dendrite length was measured using Image J software, and the numbers of spines per length of dendrite were counted along the diameter of the tertiary dendrite. About 50 measurements were taken per treatment group for all slice culture experiments, n = 50.

In vivo studies The Synb1-ELP and Synb1-ELP-PKI proteins were labeled with rhodamine or Alexa 750 maleimide labeling kit (Invitrogen) to produce fluorescent labeled Synb1-ELP peptides as described previously (Hearst et al. 2011). The Synb1-ELP polypeptide is commonly used as a control peptides for uptake studies (Massodi et al. 2009; Moktan et al. 2012), where the polypeptides are commonly labeled on the C terminal cysteine residue with a fluorescent dye (rhodamine or Alexa 750) to monitor the impact of the cell-penetrating peptide on fluorescent ELP localization and uptake in vivo and in vitro (Massodi et al. 2009; Moktan et al. 2012; Hearst et al. 2011; Bidwell et al. 2013). The 3-week-old WT mice were injected i.p. with 100 mg/kg fluorescent labeled protein or 200 lL saline as the control or via intranasal (IN) route at 50 mg/ kg in a 10 ll volume or with saline as the control as described previously (Vig et al. 2006b, 2011). For all IN experiments, the peptides were given in a total volume of 10lls in one nostril only by administrating 1 lL at a time as described previously (Vig et al. 2006b, 2011). 3 h post treatment, the animals were perfused, their brains removed, and cut for frozen sectioning as described previously and visualized on an Olympus BX60 epifluorescence microscope. (Vig et al. 2011; Bidwell et al. 2013). Animals treated with Alexa 750 labeled Synb1-ELP were killed with CO2 and their bodies, brains and other organs were scanned using the VIS Live Animal Imager (Caliper Life Sciences, Hopkinton, MA, USA). The fluorescent distribution of Synb1-ELP in the brain was measured with the IVIS Live Animal Imager using an Excitation wavelength of 745 nm and an Emission wavelength of 800 nm as described previously (Hearst et al. 2011). Plasma levels of Synb1-ELP over time were measured using the IVIS live imager with an Excitation of

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 131, 101--114

104

S. M. Hearst et al.

745 nm and an Emission of 800 nm. Three-weekold FVB mice were treated via IN route at 50 mg/kg in a 10 lL volume or with saline as the control. Plasma levels and brain levels of Synb1-ELP were measured over time at 30 min, 1, 2, 4, 8, and 18 h post IN treatment. The concentration of Synb1-ELP was calculated using a standard curve of known Synb1-ELP concentrations diluted in plasma taken from untreated mice. The brain uptake was then divided by the total weight of the brain, where most 3-week-old mice had an average brain weight of 0.45–0.50 g. The fluorescent distribution of rhodamine labeled Synb1-ELP-PKI in the brain, CSF and plasma were measured with the IVIS Live Animal Imager using an Excitation wavelength of 570 nm and an Emission wavelength of 600 nm as described previously (Hearst et al. 2011). The average uptake of Synb1-ELP-PKI peptides were measured in the brain, CSF, and plasma and compared between IN- and i.p.-treated animals 6 h post treatment, n = 5 for i.p. and n = 6 for IN experiments. Controls were treated with saline vehicle by both IN and i.p. route to reduced animal number. CSF was collected by gently inserting a capillary tube under the cerebellum and into the cisterna magna. Plasma was collected using the eye bleed method. The concentration of Synb1-ELP-PKI in CSF and plasma was calculated using a standard curve of known Synb1-ELP-PKI concentrations. Graphs were produced in Excel and show Mean  SD of the changes in Synb1-ELP-PKI levels between IN- and i.p.-treated mice. Statistics PKA activity assay experiments were run in duplicates and repeated twice. Data were analyzed using Student’s t-test, where difference between samples was considered as statistically significant at p < 0.05. The western blot experiments were repeated for a total of three times to confirm observations. Statistical analysis was done using Students t-test, where p < 0.05 is considered significant. Slice culture experiments were repeated at least three times to confirm observations. Statistically significant changes in the dendritic growth area or the number of spines were calculated with the Students t-test, where p < 0.05 was considered significant. All in vivo experiments were repeated with a total of three animals per treatment group to confirm results. In some bio-distribution experiments, up to 6 animals per time point were used. The statistical analysis for the in vivo was done using Students t-test, where p < 0.05 is considered significant.

Results The design of an ELP-based PKA inhibitory polypeptide In our previous work, we demonstrated that the ELP-based peptide carrier Synb1-ELP (Fig. 1a), has a remarkable potential to deliver therapeutics to the cerebellum (Hearst et al. 2011). In this study, we have designed a PKA inhibitory polypeptide comprising of three fused peptides: (i) the ELP, which is thermally responsive peptide that increases peptide half-life, (ii) the PKI inhibitory peptide (Fig. 1c), derived from the PKIa protein, interacts specifically with the catalytic subunit of PKA inhibiting kinase activity (Demaille et al. 1977; Glass et al. 1992), and (iii) a cell-penetrating peptide, Synb1, to enhance intracellular delivery and delivery across the blood–brain barrier (Fig. 1b). Interestingly, the PKIa protein is highly expressed in the cerebellum and localizes to PCs (Van

Patten et al. 1992; Seasholtz et al. 1995). The integrity of purified proteins was checked by; the sodium dodecyl sulfate– polyacrylamide gel electrophoresis gel stained using Comassie blue for the purified ELP peptides, Synb1-ELP and Synb1ELP-PKI (Fig. 1d). ELP-based peptides have a molecular weight around 60–65 kd (Dreher et al. 2007; Bidwell et al. 2009). Next, we tested the PKA inhibitory function of our peptide, Synb1-ELP-PKI compared to the PKI peptide. We used the PKA activity assay kit from Enzo Life Sciences (see methods). We tested the ability of Synb1-ELP-PKI to inhibit 4.0 Units of active PKA and compared that to PKI peptide alone or Synb1-ELP. We found that Synb1-ELP-PKI and the PKI peptide both significantly inhibited PKA activity at 5.0 and 10.0 lM as compared to Control (no inhibitors) (Fig. 1e). However, 10.0 lM Synb1-ELP had no inhibitory effects on PKA activity (Fig 1e and f). Next, we titrated Synb1-ELP-PKI and PKI from 0.5 lM to 20.0 lM (0.5, 1.0, 2.5, 5.0, 10.0, and 20.0 lM) and measured the PKA inhibitory effects using 4.0 Units of active PKA enzyme (Fig. 1f). We found that both Synb1-ELP-PKI and PKI inhibited PKA activity at a similar rate (Fig. 1f). Both Synb1-ELP-PKI and PKI had a similar EC50 value between 0.5 and 1.0 lM (Fig. 1f). Overall, we found that the Synb1-ELP-PKI peptide showed similar inhibition of PKA activity as compared to the PKI peptide. Synb1-ELP-PKI reduces ATXN1 aggregation and S776 phosphorylation We showed earlier that alternations in the PKA activity affected mutant ATXN1 nuclear aggregation and S776 phosphorylation (Hearst et al. 2010), whereas the mutant ATXN1 A776 protein fails to form inclusions (Chen et al. 2003; Emamian et al. 2003; Hearst et al. 2010). We further demonstrated that PKA activator, FSK, increases ATXN1 S776 phosphorylation and aggregation in HEK cells expressing GFP-ATXN1[82Q] (Hearst et al. 2010). To test the effects of our PKA inhibitory polypeptide on inclusion formation and mutant ATXN1 S776 phosphorylation, we treated stable GFP-ATXN1[82Q] HEK cells with 50 lM Synb1-ELP (the control peptide), Synb1-ELP-PKI, or 10 lM H89, PKA inhibitor. Next, the cells were washed with phosphate-buffered saline and treated with 60 lM FSK or serum-free media for 4 h and then fixed for fluorescent microscopic analysis. Fig. 2a displays the impact of the various treatment conditions on GFP-ATXN1[82Q] nuclear inclusion formation. GFP-ATNX1[82Q] expressing cells have been reported to display three phenotypes: soluble diffuse localization within the nucleus, small punctate foci or larger inclusions (Fig. 2b) (Parfitt et al. 2009; Hearst et al. 2010). Control treated cells displayed a more soluble GFPATXN1[82Q] phenotype; while cells treated with FSK or Synb1-ELP+FSK displayed large GFP-ATXN1[82Q] inclusions (Fig. 2a). Earlier, we reported inhibiting PKA with H89 reduces GFP-ATXN1[82Q] inclusions (Hearst et al. 2010). Furthermore, cells treated with either Synb1-ELP-PKI

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 131, 101--114

PKA inhibitory polypeptide to treat SCA1

(a)

105

(d)

(b)

(c) (e)

(f)

Fig. 1 Displayed is a diagram of the (a) control polypeptide carrier, Synb1-elastin-like polypeptide (ELP), and the therapeutic polypeptide (b) Synb1-ELP-PKA inhibitory peptide (PKI). Displayed are the three key elements: (i) ELP, a thermally responsive peptide; (ii) PKI peptide, a known cAMP-dependent protein kinase (PKA) inhibitory peptide; and (iii) a cell-penetrating peptide, Synb1, to enhance intracellular delivery. (c) Shown is the PKI peptide sequence: TYADFIASGRTGRRNAI. (d) Shown is an sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS–PAGE) gel Coomassie

stained for the purified ELP peptides: Synb1-ELP and Synb1-ELPPKI. ELP peptides have a molecular weight around 60 to 65kD. (e) Shown is the Mean  SD of PKA activity after incubation with inhibitors PKI and Synb1-ELP-PKI or Synb1-ELP. Statistics were performed using the Student’s t-test. *PKI or Synb1-ELP-PKI versus Control, *p < 0.05. (f) The graph displays the inhibition curve of PKA activity upon titration of PKI or Synb1-ELP-PKI from 0.5 to 20.0 lM. Shown is the Mean  SD of PKA activity after incubation with inhibitors PKI and Synb1-ELP-PKI or Synb1-ELP.

or H89 displayed a more soluble GFP-ATXN1[82Q] phenotype suggesting that Synb1-ELP-PKI may be reducing FSK-PKA-stimulated GFP-ATXN1[82Q] inclusion formation. To confirm this finding, the percentage of GFP-ATXN1 [82Q] HEK cells expressing soluble, small punctate foci or large inclusion under the various treatment conditions were calculated (Fig. 2c). FSK and Synb1-ELP+FSK stimulated cells showed a significantly higher number of large inclusions as compared with Control-treated cells; while Synb1ELP-PKI+FSK or H89 + FSK did not induce significant inclusion body formation as compared with Control-treated cells (Fig. 2c). In contrast, treatment with FSK and Synb1ELP+ FSK generated a significant number of large inclusions as compared with Synb1-ELP-PKI+ FSK treatment or H89 + FSK treatment (Fig. 2c). These data suggest that Synb1-ELP-PKI may reduce ATXN1[82Q] inclusion formation by inhibiting PKA phosphorylation of the ATXN1 S776 site. To confirm this idea, GFP-ATXN1[82Q] expressing HEK cells were placed in 6-well plates and pre-treated with 5.0, 10.0, 25.0, and 50.0 lM Synb1-ELP-PKI or 50.0 lM Synb1-ELP for 2 h and then treated with 60 lM FSK for 4 h. Again we used a known PKA inhibitor, where

cells were pre-treated with 10 lM H89 for 2 h followed by 60 lM FSK for 4 h; H89 is a known PKA inhibitor previously reported to reduce ATXN1 S776 phosphorylation (Hearst et al. 2010). After treatment completion, the cells were lysed for western blot analysis. The western blots were probed with ATXN1 Phospho S776 antibodies to detect the level of ATXN1[82Q] S776 phosphorylation and then reprobed with GFP antibodies to detect the total level of GFP-ATXN1[82Q] protein (Fig. 2d and Fig. S1a). The protein band images were then analyzed using ImageJ software to derive band optical densities. Fig. 2e shows the optical density ratios of the ATXN1 Phospho S776 to GFPATXN1[82Q] for each treatment condition. As we reported previously, FSK stimulated ATXN1 S776 phosphorylation relative to GFP-ATXN1[82Q] Control (Fig. 2d and e) (Hearst et al. 2010). Also, Synb1-ELP+FSK stimulated ATXN1 S776 phosphorylation as compared with Control. Interestingly, all Synb1-ELP-PKI+FSK treatments and H89 + FSK reduced ATXN1 S776 phosphorylation as compared to FSK or Synb1-ELP +FSK treatment (Fig. 2d and e and Supplement Fig. 1a and b). Overall these data suggest that Synb1-ELP-PKI peptide inhibits PKA-depen-

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 131, 101--114

106

S. M. Hearst et al.

(a)

(b)

(d)

(e) (c)

Fig. 2 Green fluorescent protein (GFP)-ATXN1[82Q] expressing HEK cells were placed on 2-well slides. (a) Shown is the epi-fluorescence of HEK cells expressing GFP-ATXN1[82Q] under the reported treatment conditions, where DAPI nuclear stain is shown in blue and GFPATNX1 in green, scale bar 10 lm. (b) HEK cells expressing GFPATXN1[82Q] have been reported to display three phenotypes, soluble nuclear staining, small punctate foci, and large nuclear inclusions, scale bar 10 lm. (c) The graph shows the effect of peptide treatment on forskolin (FSK) stimulated inclusion formation. The graph shows the Mean  SE of the Cell Percentage expressing the various three phenotypes: soluble nuclear staining, small punctate foci, and large nuclear inclusions. Statistics were performed using the Student’s t-test. *FSK or Synb1-elastin-like polypeptide (ELP)+FSK versus Control,

*FSK versus Synb1-ELP-PKA inhibitory peptide (PKI) + FSK, *p < 0.05. (d) GFP-ATXN1[82Q] expressing HEK cells were placed on 6 well plates and pre-treated with 5.0, 10.0, 25.0, and 50.0 lM Synb1-ELP-PKI or 50.0 lM Synb1-ELP for 2 h and then treated with 60 lM FSK for 4 h and then lysed for protein analysis. Shown is the western blot data detecting ATNX1 Phospho S776 and GFP under the described treatment conditions. The optical densities of the protein bands were taken using ImageJ software. (e) The graph shows the Mean  SD ratio of ATNX1 Phospho S776 to GFP-ATXN1[82Q] of three experimental repeats. Statistics were performed using the Student’s t-test. *FSK or Synb1-ELP+FSK versus Control, *FSK or Synb1-ELP+FSK versus all Synb1-ELP-PKI + FSK treatments, *p < 0.05.

dent mutant ATXN1[82Q] S776 phosphorylation and inclusion formation.

Synb1-ELP was labeled with the florescent dye rhodamine (Synb1-ELP-Rho) as described previously (Bidwell and Raucher 2005; Bidwell et al. 2010, 2012, 2013). Cerebellar slices were prepared from 9-day-old mice WT mice and treated with 10.0 lM Synb1-ELP-Rho for 16 h. The sections where fixed and then stained with antibodies for Bergmann glia specific protein S100B (Fig. 3a) or PC specific protein

Synb1-ELP-PKI improves SCA1 Purkinje cell morphology in cerebellar slice cultures Next, we explored the localization of Synb1-ELP in our cerebellar slice cultures (Hearst et al. 2010; Vig et al. 2011).

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 131, 101--114

PKA inhibitory polypeptide to treat SCA1

107

(c)

(a)

(b)

(d)

(e)

(f)

Fig. 3 Cerebellar slices from WT mice were treated with 10.0 lM Rhodamine labeled Synb1-elastin-like polypeptide (ELP) (Synb1-ELPRho). (a) Shown is the Synb1-ELP-Rho localization in red and S100B staining in green from WT cerebellar cultures, scale bar 10 lm. (b) Shown is the Synb1-ELP-Rho localization in red and calbindin (CaB) staining in green from WT cerebellar cultures, scale bar 10 lm. (c) Shown is the Synb1-ELP-Rho localization in red and DAPI stain in blue over the allotted time from WT cerebellar cultures, scale bar 10 lm. Next, green fluorescent protein (GFP)-WT or GFP-SCA1 slices were treated with media as Control, and various concentrations of Synb1-ELP, Synb1-ELP-TRTK or Synb1-ELP-PKA inhibitory

peptide (PKI) every 48 h for 6 to 10 days. (d) Shown is the Mean  SD of GFP-WT Purkinje cell growth area measured using ImageJ software. The dotted line demonstrates the Control base line. (e) Shown is the Mean  SD of GFP-SCA1 Purkinje cell growth area measured using ImageJ software. The dotted line demonstrates the Control base line. Statistics were performed using the Student’s t-test. *Synb1-ELP-TRTK or Synb1-ELP-PKI versus Control, and *Synb1ELP-TRTK or Synb1-ELP-PKI versus Synb1-ELP, *p < 0.05. (f) Shown is the GFP-SCA1 Purkinje cell growth observed at 4X after Control, Synb1-ELP, Synb1-ELP-TRTK, and Synb1-ELP-PKI treatments, scale bar 10 lm.

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 131, 101--114

108

S. M. Hearst et al.

CaB (Fig. 3b). Under the 16 h treatment, Synb1-ELP-Rho highly localized to large cells that were negative for S100B (Fig. 3a) and positive for PC specific protein CaB (Fig. 3b). Thus, we concluded that the Synb1-ELP carry could be used to target PCs in slice cultures. Next, we looked at Synb1ELP-Rho localization in slice cultures over time. The WT slices treated with 10.0 lM Synb1-ELP-Rho for 4, 8, and 24 h (Fig. 3c) showed Synb1-ELP-Rho localization in PCs as early as 4 h and continued fluorescence uptake was seen after 8 h and 24 h treatments (Fig. 3c). Next, we tested the toxicity and therapeutic properties of our control peptide, Synb1-ELP, our therapeutic peptide, Synb1-ELP-PKI, and another therapeutic peptide, Synb1-ELP-TRTK to WT and SCA1 PC slice cultures. Synb1-ELP-TRTK is another therapeutic peptide we designed previously to inhibit S100B mediated neurodegeneration in SCA1 (Hearst et al. 2011). Synb1-ELP-TRTK contains the S100B inhibitory peptide, TRTK12, developed from the high specificity S100B binding protein CapZ (Charpentier et al. 2010). GFP-WT and GFP-SCA1 cerebellar slices were prepared from 9-day-old GFP-WT mice or GFP-SCA1 mice that express the GFP protein in PCs under the PC specific gene promoter pcp2 (Vig et al. 2011). GFP-WT slices were used to test the toxicity of our peptides at a high dose. WT slices were treated with media as Control, 10.0 lM Synb1-ELP, Synb1-ELP-TRTK or Synb1-ELP-PKI every 48 h for 10 days. The GFP slices were stained using a GFP antibody and visualized using florescent microscopy. The digitized images were used to measure the change in PC dendritic area as described previously (Vig et al. 2011). We saw no significant decrease in WT PC dendritic branching or impeded growth with the high doses of our peptides as compared to Control-treated slices (Fig. 3d). Therefore, we concluded that our peptides were not toxic to PCs at the high dose studied. GFP-SCA1 PCs have been shown to have a reduced PC dendritic branch area as compared to GFP-WT PCs in slice cultures (Vig et al. 2011). Next, we tested the curative potential of our therapeutic peptides to GFP-SCA1 PC slices. GFP-SCA1 slices were treated with media as Control or 2.5 lM and 5.0 lM Synb1-ELP, Synb1-ELPTRTK or Synb1-ELP-PKI every 48 h for 6 days. We saw significant improvements in PC dendritic branch area in GFP-SCA1 slices treated with Synb1-ELP-TRTK or Synb1ELP-PKI as compared to slices treated with media as Control or 2.5 lM or as compared to 5.0 lM Synb1-ELP (Fig. 3e). Synb1-ELP-TRTK and Synb1-ELP-PKI treated SCA1 PCs showed large branching dendritic trees as compared to Control and as compared to Synb1-ELP treated slices (Fig. 3f). The fact that Synb1-ELP-PKI demonstrated a significant effect by increasing PC dendritic branching suggests that inhibiting PKA activity may be therapeutic to SCA1 PCs. We further analyzed the effect of our therapeutic peptides on tertiary dendritic spine growth in GFP-SCA1 PC slice cultures. We saw an increase in tertiary dendritic spine

formation with Synb1-ELP-TRTK and Synb1-ELP-PKI treatments as compared to Control or as compared to Synb1-ELP treatment (Fig. 4a). Also, we compared the GFP-WT PCs spines with the spines of treated GFP-SCA1 PCs. Tertiary dendritic spines of GFP-WT and GFP-SCA1 PCs treated with Synb1-ELP-TRTK or Synb1-ELP-PKI polypeptides displayed a bushy-like appearance as compared to Control and Synb1-ELP treated GFP-SCA1 PCs. To better signify this effect, we enlarged a representative tertiary dendrite from a GFP-WT and GFP-SCA1 PC under the various treatment conditions and converted the image to a binary image (Fig. 4b). We found that Synb1-ELP-TRTK or Synb1-ELP-PKI treated GFP-SCA1 PCs displayed a WT like phenotype as compared to Control and as compared to Synb1-ELP treated GFP-SCA1 PCs (Fig. 4b). Next, we quantified the number of spines per micron around the diameter of tertiary dendrites from GFP-WT and GFP-SCA1 PCs under the various treatment conditions (Fig. 4c). Synb1ELP-TRTK and Synb1-ELP-PKI treatment significantly increased the number of spines per micron on tertiary dendrite as compared to Control and as compared to Synb1ELP treated GFP-SCA1 PCs (Fig. 4c), which was similar to the number of tertiary dendritic spines per micron present in GFP-WT PCs (Fig. 4c). Control and Synb1-ELP treated GFP-SCA1 PCs had 6 to 7 spines per micron, while Synb1ELP-TRTK and Synb1-ELP-PKI treated GFP-SCA1 PCs had around 10 to 12 spines per micron; and GFP-WT PCs had around 12 to 14 spines per micron (Fig. 4c). Overall both Synb1-ELP-TRTK and Synb1-ELP-PKI displayed therapeutic effects and increased SCA1 PC dendritic branching as well as tertiary dendritic spine density. Intranasal delivery of the Synb1-ELP peptide carrier to the brain Previously, we demonstrated that the IN route is an effective peptide delivery route to treat cerebellar disorders, like SCA1 (Vig et al. 2006b, 2011). We also reported that the peptide carrier, Synb1-ELP, can be delivered to the brain via i.p. injection (Hearst et al. 2011). However, our peptide’s localization to the brain at a cellular level has never been assessed. Therefore, we tested the IN and i.p. brain delivery capabilities of our peptide carrier Synb1-ELP and examine the cerebellar uptake of our Synb1-ELP carrier at a cellular level. We treated 3-week-old WT mice with rhodamine labeled Synb1-ELP via IN and i.p. and used saline as the control. The animals were perfused 3 h post treatment and their brains removed for frozen sectioning and visualization under the fluorescent microscope. Both IN (Fig. 5a) and i.p. (Fig. 5b) routes effectively delivered the Synb1-ELP carrier to the cerebellum. We saw strong localization of Synb1-ELP in the molecular layer, the PC layer and slightly weaker localization in the granular cell layer. These data indicate that both IN and i.p. may be possible routes to deliver our therapeutic peptides to the SCA1 cerebellum. We also looked

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 131, 101--114

PKA inhibitory polypeptide to treat SCA1

(a)

109

(b)

(c)

Fig. 4 Green fluorescent protein (GFP)-SCA1 slices were treated with media as Control or 2.5 lM and 5.0 lM Synb1-elastin-like polypeptide (ELP), Synb1-ELP-TRTK or Synb1-ELP-PKA inhibitory peptide (PKI) every 48 h for 6 days. GFP-WT slices were treated with media for 6 days. (a) Shown is the GFP fluorescence of Purkinje cell (PC) spines on tertiary dendrites from GFP-WT or GFP-SCA1 slices treated with Control, Synb1-ELP, Synb1-ELPTRTK or Synb1-ELP-PKA inhibitory peptide (PKI), scale bar 1.0 lm. (b) Shown is the binary image of spines from a representative tertiary dendrite from GFP-WT or GFP-SCA1 PCs treated with

Control, Synb1-ELP, Synb1-ELP-TRTK or Synb1-ELP-PKI, scale bar 0.5 lm. (c) Tertiary dendrite length was measured using ImageJ software, and the numbers of spines per length of dendrite were counted along the diameter. The graph shows the Mean  SD of the Number of Spines per Micron of tertiary dendrite from WT or SCA1 PCs treated with Control, Synb1-ELP, Synb1-ELP-TRTK or Synb1-ELP-PKI. Statistics were taken using the Student’s t-test where p < 0.05 is considered significant. *Control versus Synb1-ELP-TRTK or Synb1-ELP-PKI, and *Synb1-ELP-TRTK or Synb1-ELP-PKI versus Synb1-ELP, *p < 0.05.

at localization of rhodamine labeled Synb1-ELP, via IN, throughout the brain and found localization in the olfactory bulb, the cerebral cortex, the hippocampus, and again the cerebellum (Fig. 5c). The olfactory bulb showed high localization of Synb1-ELP in the form of aggregates after IN administration (Fig. 5c, 409). The olfactory bulb is the closest brain tissue to the nasal passage and alongside the trigeminal pathway provides a route for IN peptide delivery to other regions of the brain (Renner et al. 2012). I.P. injections of rhodamine labeled Synb1-ELP showed similar localization to other regions of the brain as compared to IN (data not shown). Our earlier report indicates that IN route has rapid absorption (Vig et al. 2006b), therefore we chose to analyzed brain uptake over time by scanning the brains starting at 30 min post treatment in these experiments. We

treated 3 week old WT mice with Alexa 750 labeled Synb1-ELP via the IN route at 50 mg/kg and used the IVIS Live Imager to scan their brains 30 min, 1, 2, 3, 4, 8 and 18 hs post treatment. Interestingly, in the body scan, Synb1ELP was highly localized to the nasal cavity after IN treatment (Fig. 6a). We also saw some uptake of Synb1-ELP to what appeared to be the stomach after IN treatment (Fig. 6a). It is not uncommon for small amounts of IN delivered agents to be swallowed into the stomach (Southam et al. 2002). The brain scans revealed Synb1-ELP uptake to the brain post IN treatment as compared to control (Fig. 6a). As seen earlier, we saw a high localization of Synb1-ELP to the olfactory bulbs (Fig. 6a). Next, we measured the kinetic uptake of Synb1-ELP carrier to the brain after IN treatment. Fig. 6b displays the total brain uptake of Synb1-ELP as lg of

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 131, 101--114

110

S. M. Hearst et al.

(a)

(c)

(b)

Fig. 5 3-week-old WT mice were given Synb1-elastin-like polypeptide (ELP)-Rho via intranasal (IN) or i.p. route and saline as the Control. (a) Shown are cerebellar sections from IN-treated mice. Shown in red is the localization of Synb1-ELP in the cerebellum and DAPI nuclear stain in blue, left scale bar 10 lm, right scale bar 25 lm. (b) Shown are cerebellar sections from i.p.-treated mice.

Shown in red is the localization of Synb1-ELP in the cerebellum and DAPI nuclear stain in blue, left scale bar 10 lm, right scale bar 25 lm. (c) Shown in red is the localization of Synb1-ELP in the olfactory bulb, the cerebral cortex, the hippocampus, and the cerebellum and DAPI nuclear stain in blue, left scale bar 25 lm (209), right scale bar 10 lm (409).

Synb1ELP per gram of brain tissue over time. Synb1-ELP peaks the brain 30 min after treatment and then drops at 2 h and then increases again at 4 h (Fig. 6b). The rise in brain levels seen at 4 h post treatment is most likely because of the redelivery of Synb1-ELP to the brain via the blood stream. Next, we quantified the uptake in different regions of the brain by taking an ROI of the olfactory bulb, cerebral cortex and the cerebellum over time as described previously (Hearst et al. 2011). We found that Synb1-ELP was highly localized to the olfactory bulb and peaked at 30 min (Fig. 6c). Synb1ELP peaked the cerebellum and cerebral cortex at 4 h, which may be because of both the trigeminal pathway delivery and blood stream delivery of Synb1-ELP to these regions of the brain (Fig. 6c). To ensure that our peptide Synb1-ELP-PKI had similar IN and i.p. delivery capabilities as the Synb1ELP carrier, we labeled the Synb1-ELP-PKI peptide with rhodamine and compared the brain distribution, CSF level, and plasma level 6 h after IN or i.p. administration of 100 mg/kg Synb1-ELP-PKI-Rho. We found that Synb1ELP-PKI localized to the brain in both IN- and i.p.-treated animals (Fig. 7a). However, i.p.-treated mice showed higher

brain uptake as compared to IN-treated mice (Fig. 7a and b). Also, CSF and plasma levels were higher in i.p.-treated mice as compared to IN-treated mice (Fig. 7c and d). Although, the i.p. route delivered more peptide to the brain, it had a lower CSF to plasma ratio as compared to the IN route (Fig. 7e). Our data suggest that IN may be the better delivery route to target the Synb1-ELP-PKI peptide to the brain while limiting its delivery to the blood stream and non-targeted organs and tissues.

Discussion SCA1 is a neurological CAG repeat disorder caused by a mutation in the ATXN1 gene (Koeppen 2005; Orr and Zoghbi 2007; Matilla-Duenas et al. 2008). This mutation causes a polyglutamine expanded form of the ATXN1 protein which is primarily toxic to PCs of the cerebellum as well as other neurons throughout the brain (Koeppen 2005; Orr and Zoghbi 2007; Matilla-Duenas et al. 2008). Overexpression of the human mutant ATXN1 gene in PCs of SCA1 transgenic (Tg) mice results in a disease state and ataxia very similar to SCA1

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 131, 101--114

PKA inhibitory polypeptide to treat SCA1

(a)

111

(b)

(c)

Fig. 6 3-week-old WT mice were given Alexa-750 labeled Synb1elastin-like polypeptide (ELP) peptide via intranasal (50 mg/kg). 3 h post treatment their bodies and then their brains were scanned with the IVIS imager as previously described (Hearst et al. 2011). (a) The images show the localization of Synb1-ELP after intranasal administration. The Color Scale shows Epi-fluorescence in Counts from 0 to

4000 where green is a high level of Synb1-ELP localization, red is a low level, and blue is background fluorescence. Scale bars 4 mm. (b) Shown is the Mean brain uptake or Mean plasma level of Alexa-750 labeled Synb1-ELP peptide via intranasal (50 mg/kg) over time. (c) Shown is the Mean localization of Synb1-ELP in the olfactory bulb, cerebral cortex, and cerebellum over time.

patients (Burright et al. 1995). Much research has focused on the mechanisms that lead to mutant ATXN1-mediated PC neurodegeneration with major emphasis on the phosphorylation of S776 on the mutant ATXN1 protein (Chen et al. 2003; Emamian et al. 2003; Jorgensen et al. 2009; Hearst et al. 2010). The mutant ATXN1 S776 once phosphorylated is stabilized by binding of the 14-3-3 protein to resist protein degradation (Chen et al. 2003). Many animal studies have demonstrated the significance of S776 phosphorylation and its effect on PC degeneration and ataxic behavioral deficits. In this study, we have designed a PKA inhibitory peptide to target mutant ATNX1 S776 phosphorylation as a potential SCA1 therapeutic. Our therapeutic peptide was designed to take advantage of the unique properties of each of its three fused peptides: (i) the ELP, a thermally responsive polypeptide, (ii) the PKI peptide, a known PKA inhibitory peptide, (iii) a cell-penetrating peptide, Synb1, to enhance intracellular delivery (Fig. 1b). The Synb1 peptide, derived from protegrin peptides, has been shown to mediate transport across the blood–brain barrier (Rousselle et al. 2000; Sarantseva et al. 2009). On the C-terminus, we fused the PKA inhibitory peptide, PKI, to our therapeutic peptide. The PKI peptide is derived from the PKIa protein which specifically inhibits PKA activity through binding the PKA catalytic subunit (Demaille et al. 1977; Glass et al. 1992). Three endogenous PKI isoforms (PKIa, PKIb, PKIc) have been identified from

distinct genes expressed in a wide range of tissues from the brain to the testis (Olsen and Uhler 1991; Collins and Uhler 1997; Zheng et al. 2000). PKIa is expressed in skeletal and cardiac muscle as well as the brain and is highly expressed in cerebellar PCs (Van Patten et al. 1992; Seasholtz et al. 1995). We believe that using the Synb1-ELP-PKI peptide to block PKA activity in PCs may be therapeutically beneficial in SCA1. In our PKA activity assay, we found that the Synb1ELP-PKI peptide showed similar inhibition of PKA activity as compared to the PKI peptide alone (Fig. 1). These data suggest that the addition of the Synb1-ELP to form the Synb1-ELP-PKI peptide does not interfere with the inhibitory function of the PKI peptide. Our in vitro studies concluded that our therapeutic peptide, Synb1-ELP-PKI, functions as PKA inhibitor to combat PKA-mediated mutant ATNX1 S776 phosphorylation and aggregation. The mutant ATXN1 protein makes nuclear inclusions both in cell culture and mouse models (Chen et al. 2003; Jorgensen et al. 2009; Hearst et al. 2010). In cell culture, ATXN1 S776 phosphorylation causes inclusion formation, whereas the mutant ATXN1 A776 protein fails to form inclusions (Chen et al. 2003; Emamian et al. 2003; Hearst et al. 2010). Previously, we demonstrated that PKA activator, FSK, increases mutant ATXN1 inclusion formation and ATXN1 S776 phosphorylation in HEK cells expressing GFP-ATXN1[82Q] (Hearst et al. 2010). Our cell culture studies showed that the Synb1-

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 131, 101--114

112

S. M. Hearst et al.

(a)

(b)

(c)

(d)

(e)

Fig. 7 WT mice were given rhodamine labeled Synb1-elastin-like polypeptide (ELP)-PKA inhibitory peptide (PKI) peptide via intranasal (IN) or i.p. (100 mg/kg). 6 h post treatment their CSF and plasma were taken and then their brains were removed and scanned with the IVIS imager as previously described (Hearst et al. 2011). (a) The images show the localization of Synb1-ELP-PKI in the brain after IN

and i.p. administration. Scale bars 4 mm. (b) Shown is the Mean  SD brain uptake (c) Mean  SD CSF level and (d) Mean  SD plasma level of rhodamine labeled Synb1-ELP-PKI peptide taken from IN and i.p.-treated mice. (e) This graph shows the Mean  SD of the CSF to plasma ratio comparing IN and i.p.-treated mice. i.p.: n = 5, IN: n = 6.

ELP-PKI peptide inhibits PKA-dependent GFP-ATXN1 [82Q] inclusion formation and ATXN1 S776 phosphorylation in HEK cells (Fig. 2). Furthermore, we show that our therapeutic peptide can be delivered to PCs in cerebellar slice cultures (Fig. 3). Also, we show that treating SCA1 PCs with our therapeutic peptide, Synb1-ELP-PKI, improved PC dendritic growth (Fig. 3) as well as improved PC tertiary dendritic spine formation (Fig. 4). Taken together, our data support the use of Synb1-ELP-PKI peptide as a possible SCA1 therapeutic. The first and foremost morphological change seen in SCA1 Tg mice is the development of cytoplasmic PC vacuoles containing the Bergmann glial protein S100B (Skinner et al. 2001; Vig et al. 2006a, 2009). Our previous work has demonstrated that S100B expression is upregulated in SCA1 mice and that inhibiting S100B via TRTK12 peptide therapy reduces SCA1 behavioral deficits (Vig et al. 2011). S100B is a known RAGE ligand, and stimulation of S100B-RAGE signaling increases the production of reactive oxygen species, causing oxidative damage to neurons (Adami et al. 2004; Donato et al. 2009; Sorci et al. 2010; Hearst et al. 2011). Therefore, we designed an ELPbased S100B inhibitory peptide, Synb1-ELP-TRTK, and tested its therapeutic effects in vitro. We reported that S100B

induced oxidative damage to neurons can be blocked by Synb1-ELP-TRTK treatment (Hearst et al. 2011). In this study, we further explore the therapeutic potential of Synb1ELP-TRTK and found that inhibiting S100B via Synb1-ELPTRTK treatment improves SCA1 PC dendritic growth (Fig. 3) and increase SCA1 PC tertiary dendritic spine formation (Fig. 4). Our previous work has shown that inhibiting S100B using the TRTK12 peptide had similar effects in GFP-SCA1 slice cultures, where TRTK12 treatment significantly increased dendritic branching (Vig et al. 2011). It is no surprise that the TRTK12-based peptide, Synb1-ELP-TRTK, would also show similar therapeutic effects. These data along with our previous work support the Synb1-ELP-TRTK peptide as a possible SCA1 therapeutic. ELP-based technology has a great potential to thermally target therapeutics to the cerebellum and possibly other areas of the brain to combat neurodegenerative disorders (Hearst et al. 2011). Our previous work showed that using focused hyperthermia to raise the temperature of the cerebellum by 2°C was sufficient to thermally target our peptide carrier, Synb1-ELP (Fig. 1a), to the mouse cerebellum (Hearst et al. 2011). In this study, we further analyzed the localization of our peptide carrier in vivo via a new route. Here, we report the IN route as a new brain delivery strategy for ELP-based

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 131, 101--114

PKA inhibitory polypeptide to treat SCA1

peptides. IN is becoming a popular drug delivery route used to treat neurodegenerative diseases because of its practical and non-invasive way of bypassing the blood–brain barrier to target the brain (Hanson and Frey 2008; Campbell et al. 2012; Renner et al. 2012). We demonstrated that both i.p. and IN administration localized the Synb1-ELP carrier and Synb1-ELP-PKI to the cerebellum and other regions of the brain. Overall, our in vitro studies concluded that our peptides, Synb1-ELP-PKI and Synb1-ELP-TRTK, are potential therapeutic candidate for further in vivo studies in SCA1 mice. Also, we compared the i.p. route to the IN route and found that IN-treated mice have a higher CSF to plasma ratio. These data suggest that IN may be a better delivery route to target Synb1-ELP peptides to the brain while reducing uptake in the blood stream, which may contribute to uptake in non-targeted organs and tissues. However, the IN route has volume restrictions that can limit the dose of peptide used for treatment (Southam et al. 2002). In our future work, we will measure the ability of the Synb1-ELP carrier to deliver the PKI and the TRTK peptides to PCs in vivo as well as assess the therapeutic effectiveness of Synb1-ELP-PKI or -TRTK to suppress SCA1 phenotype in the mouse models. Our Synb1-ELP carrier is not just limited to peptides, but could be modified to accommodate drugs, siRNA or DNA to deliver these therapeutics to the cerebellum and other regions of the brain.

Acknowledgments and conflict of interest disclosure This work was funded by National Institutes of Health. The data reported in this manuscript have not been published or submitted for publication elsewhere. All authors have agreed to the contents of this article and there are no ethical issues involved. All experiments were conducted in compliance with the ARRIVE guidelines. The authors have no conflict of interest to declare.

Supporting information Additional supporting information may be found in the online version of this article at the publisher's web-site: Figure S1. GFP-ATXN1[82Q] expressing HEK cells were placed on 6-well plates and pre-treated with 50.0 lM Synb1-ELPPKI or 50.0 lM Synb1-ELP of 50.0 lM H89 for 2 h and then treated with 60 lM FSK for 4 h and then lysed for protein analysis.

References Adami C., Bianchi R., Pula G. and Donato R. (2004) S100B-stimulated NO production by BV-2 microglia is independent of RAGE transducing activity but dependent on RAGE extracellular domain. Biochim. Biophys. Acta 1742, 169–177. Allen T. M. (1998) Liposomal drug formulation: rationale for development and what we can expect in the future. Drugs 56, 747–756.

113

Bidwell G. L., 3rd and Raucher D. (2005) Application of thermally responsive polypeptides directed against c-Myc transcriptional function for cancer therapy. Mol. Cancer Ther. 4, 1076–1085. Bidwell G. L., 3rd and Raucher D. (2010) Cell penetrating elastin-like polypeptides for therapeutic peptide delivery. Adv. Drug Deliv. Rev. 62, 1486–1496. Bidwell G. L., 3rd, Davis A. N. and Raucher D. (2009) Targeting a cMyc inhibitory polypeptide to specific intracellular compartments using cell penetrating peptides. J. Control. Release 135, 2–10. Bidwell G. L. 3rd, Whittom A. A., Thomas E., Lyons D., Hebert M. D. and Raucher D. (2010) A thermally targeted peptide inhibitor of symmetrical dimethylation inhibits cancer-cell proliferation. Peptides 31, 834–841. Bidwell G. L., 3rd, Perkins E. and Raucher D. (2012) A thermally targeted c-Myc inhibitory polypeptide inhibits breast tumor growth. Cancer Lett. 319, 136–143. Bidwell G. L., 3rd, Perkins E., Hughes J., Khan M., James J. R. and Raucher D. (2013) Thermally targeted delivery of a c-Myc inhibitory polypeptide inhibits tumor progression and extends survival in a rat glioma model. PLoS ONE 8, e55104. Burright E. N., Clark H. B., Servadio A., Matilla T., Feddersen R. M., Yunis W. S., Duvick L. A., Zoghbi H. Y. and Orr H. T. (1995) SCA1 transgenic mice: a model for neurodegeneration caused by an expanded CAG trinucleotide repeat. Cell 82, 937–948. Campbell C., Morimoto B. H., Nenciu D. and Fox A. W. (2012) Drug development of intranasally delivered peptides. Ther. Deliv. 3, 557–568. Charpentier T. H., Thompson L. E., Liriano M. A., Varney K. M., Wilder P. T., Pozharski E., Toth E. A. and Weber D. J. (2010) The effects of CapZ peptide (TRTK-12) binding to S100B-Ca2+ as examined by NMR and X-ray crystallography. J. Mol. Biol. 396, 1227–1243. Chen H. K., Fernandez-Funez P., Acevedo S. F. et al. (2003) Interaction of Akt-phosphorylated ataxin-1 with 14-3-3 mediates neurodegeneration in spinocerebellar ataxia type 1. Cell 113, 457–468. Collins S. P. and Uhler M. D. (1997) Characterization of PKIgamma, a novel isoform of the protein kinase inhibitor of cAMP-dependent protein kinase. J. Biol. Chem. 272, 18169–18178. Demaille J. G., Peters K. A. and Fischer E. H. (1977) Isolation and properties of the rabbit skeletal muscle protein inhibitor of adenosine 3’,5’-monophosphate dependent protein kinases. Biochemistry 16, 3080–3086. Donato R., Sorci G., Riuzzi F., Arcuri C., Bianchi R., Brozzi F., Tubaro C. and Giambanco I. (2009) S100B’s double life: intracellular regulator and extracellular signal. Biochim. Biophys. Acta 1793, 1008–1022. Dreher M. R., Liu W., Michelich C. R., Dewhirst M. W. and Chilkoti A. (2007) Thermal cycling enhances the accumulation of a temperature-sensitive biopolymer in solid tumors. Cancer Res. 67, 4418–4424. Emamian E. S., Kaytor M. D., Duvick L. A., Zu T., Tousey S. K., Zoghbi H. Y., Clark H. B. and Orr H. T. (2003) Serine 776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice. Neuron 38, 375–387. Glass D. B., Feller M. J., Levin L. R. and Walsh D. A. (1992) Structural basis for the low affinities of yeast cAMP-dependent and mammalian cGMP-dependent protein kinases for protein kinase inhibitor peptides. Biochemistry 31, 1728–1734. Hanson L. R. and Frey W. H. 2nd (2008) Intranasal delivery bypasses the blood-brain barrier to target therapeutic agents to the central nervous system and treat neurodegenerative disease. BMC Neurosci. 9 Suppl 3, S5.

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 131, 101--114

114

S. M. Hearst et al.

Hearst S. M., Lopez M. E., Shao Q., Liu Y. and Vig P. J. (2010) Dopamine D2 receptor signaling modulates mutant ataxin-1 S776 phosphorylation and aggregation. J. Neurochem. 114, 706–716. Hearst S. M., Walker L. R., Shao Q., Lopez M., Raucher D. and Vig P. J. (2011) The design and delivery of a thermally responsive peptide to inhibit S100B-mediated neurodegeneration. Neuroscience 197, 369–380. Jorgensen N. D., Andresen J. M., Lagalwar S. et al. (2009) Phosphorylation of ATXN1 at Ser776 in the cerebellum. J. Neurochem. 110, 675–686. Kissel M., Peschke P., Subr V., Ulbrich K., Schuhmacher J., Debus J. and Friedrich E. (2001) Synthetic macromolecular drug carriers: biodistribution of poly[(N-2-hydroxypropyl)methacrylamide] copolymers and their accumulation in solid rat tumors. PDA J. Pharm. Sci. Technol. 55, 191–201. Koeppen A. H. (2005) The pathogenesis of spinocerebellar ataxia. Cerebellum 4, 62–73. Liu W., Dreher M. R., Furgeson D. Y., Peixoto K. V., Yuan H., Zalutsky M. R. and Chilkoti A. (2006) Tumor accumulation, degradation and pharmacokinetics of elastin-like polypeptides in nude mice. J. Control. Release 116, 170–178. Liu Y., Buck D. C. and Neve K. A. (2008) Novel interaction of the dopamine D2 receptor and the Ca2+ binding protein S100B: role in D2 receptor function. Mol. Pharmacol. 74, 371–378. Massodi I., Thomas E. and Raucher D. (2009) Application of thermally responsive elastin-like polypeptide fused to a lactoferrin-derived peptide for treatment of pancreatic cancer. Molecules 14, 1999– 2015. Matilla-Duenas A., Goold R. and Giunti P (2008) Clinical, genetic, molecular, and pathophysiological insights into spinocerebellar ataxia type 1. Cerebellum 7, 106–114. McPherson D. T., Morrow C., Minehan D. S., Wu J., Hunter E. and Urry D. W. (1992) Production and purification of a recombinant elastomeric polypeptide, G-(VPGVG)19-VPGV, from Escherichia coli. Biotechnol. Prog. 8, 347–352. Meyer D. E. and Chilkoti A. (1999) Purification of recombinant proteins by fusion with thermally-responsive polypeptides. Nat. Biotechnol. 17, 1112–1115. Moktan S., Ryppa C., Kratz F. and Raucher D. (2012) A thermally responsive biopolymer conjugated to an acid-sensitive derivative of paclitaxel stabilizes microtubules, arrests cell cycle, and induces apoptosis. Invest. New Drugs 30, 236–248. Neve K. A., Seamans J. K. and Trantham-Davidson H. (2004) Dopamine receptor signaling. J. Recept. Signal Transduct. Res. 24, 165–205. Olsen S. R. and Uhler M. D. (1991) Isolation and characterization of cDNA clones for an inhibitor protein of cAMP-dependent protein kinase. J. Biol. Chem. 266, 11158–11162. Ong S. R., Trabbic-Carlson K. A., Nettles D. L., Lim D. W., Chilkoti A. and Setton L. A. (2006) Epitope tagging for tracking elastin-like polypeptides. Biomaterials 27, 1930–1935. Orr H. T. and Zoghbi H. Y. (2007) Trinucleotide repeat disorders. Annu. Rev. Neurosci. 30, 575–621. Parfitt D. A., Michael G. J., Vermeulen E. G., Prodromou N. V., Webb T. R., Gallo J. M., Cheetham M. E., Nicoll W. S., Blatch G. L. and Chapple J. P. (2009) The ataxia protein sacsin is a functional co-chaperone that protects against polyglutamine-expanded ataxin1. Hum. Mol. Genet. 18, 1556–1565.

Renner D. B., Svitak A. L., Gallus N. J., Ericson M. E., Frey W. H., 2nd and Hanson L. R. (2012) Intranasal delivery of insulin via the olfactory nerve pathway. J. Pharm. Pharmacol. 64, 1709– 1714. Rousselle C., Clair P., Lefauconnier J. M., Kaczorek M., Scherrmann J. M. and Temsamani J. (2000) New advances in the transport of doxorubicin through the blood-brain barrier by a peptide vectormediated strategy. Mol. Pharmacol. 57, 679–686. Sarantseva S. V., Bol’shakova O. I., Timoshenko S. I., Kolobov A. A., Vitek M. P. and Shvartsman A. L. (2009) Protein transduction domain peptide mediates delivery to the brain via the blood-brain barrier in Drosophila. Biomed. Khim. 55, 41–49. Seasholtz A. F., Gamm D. M., Ballestero R. P., Scarpetta M. A. and Uhler M. D. (1995) Differential expression of mRNAs for protein kinase inhibitor isoforms in mouse brain. Proc. Natl Acad. Sci. USA 92, 1734–1738. Skinner P. J., Vierra-Green C. A., Clark H. B., Zoghbi H. Y. and Orr H. T. (2001) Altered trafficking of membrane proteins in purkinje cells of SCA1 transgenic mice. Am. J. Pathol. 159, 905–913. Sorci G., Bianchi R., Riuzzi F., Tubaro C., Arcuri C., Giambanco I. and Donato R. (2010) S100B Protein, a damage-associated molecular pattern protein in the brain and heart, and beyond. Cardiovasc. Psychiatry Neurol. doi: 10.1155/2010/656481. [epub ahead of print]. Southam D. S., Dolovich M., O’Byrne P. M. and Inman M. D. (2002) Distribution of intranasal instillations in mice: effects of volume, time, body position, and anesthesia. Am. J. Physiol. Lung Cell. Mol. Physiol. 282, L833–L839. Urry D. W. (1992) Free energy transduction in polypeptides and proteins based on inverse temperature transitions. Prog. Biophys. Mol. Biol. 57, 23–57. Van Patten S. M., Howard P., Walsh D. A. and Maurer R. A. (1992) The alpha- and beta-isoforms of the inhibitor protein of the 3’,5’-cyclic adenosine monophosphate-dependent protein kinase: characteristics and tissue- and developmental-specific expression. Mol. Endocrinol. 6, 2114–2122. Vig P. J., Lopez M. E., Wei J., D’Souza D. R., Subramony S., Henegar J. and Fratkin J. D. (2006a) Glial S100B positive vacuoles in purkinje cells: earliest morphological abnormality in SCA1 transgenic mice. J. Neurol. Sci. Turk. 23, 166–174. Vig P. J., Subramony S. H., D’Souza D. R., Wei J. and Lopez M. E. (2006b) Intranasal administration of IGF-I improves behavior and Purkinje cell pathology in SCA1 mice. Brain Res. Bull. 69, 573– 579. Vig P. J., Shao Q., Subramony S. H., Lopez M. E. and Safaya E. (2009) Bergmann glial S100B activates myo-inositol monophosphatase 1 and Co-localizes to purkinje cell vacuoles in SCA1 transgenic mice. Cerebellum 8, 231–244. Vig P. J., Hearst S., Shao Q., Lopez M. E., Murphy H. A., 2nd and Safaya E. (2011) Glial S100B protein modulates mutant ataxin-1 aggregation and toxicity: TRTK12 peptide, a potential candidate for SCA1 therapy. Cerebellum 10, 254–266. Zheng L., Yu L., Tu Q., Zhang M., He H., Chen W., Gao J., Yu J., Wu Q. and Zhao S. (2000) Cloning and mapping of human PKIB and PKIG, and comparison of tissue expression patterns of three members of the protein kinase inhibitor family, including PKIA. Biochem. J. 349, 403–407.

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 131, 101--114