VGF function in depression and antidepressant efficacy.

Brain-derived neurotrophic factor (BDNF) is a critical effector of depression-like behaviors and antidepressant responses. Here, we show that VGF (non-acronymic), which is robustly regulated by BDNF/TrkB signaling, is downregulated in hippocampus (male/female) and upregulated in nucleus accumbens (NAc) (male) in depressed human subjects and in mice subjected to chronic social defeat stress (CSDS). Adeno-associated virus (AAV)-Cre-mediated Vgf ablation in floxed VGF mice, in dorsal hippocampus (dHc) or NAc, led to pro-depressant or antidepressant behaviors, respectively, while dHc- or NAc-AAV-VGF overexpression induced opposite outcomes. Mice with reduced VGF levels in the germ line (Vgf+/-) or in dHc (AAV-Cre-injected floxed mice) showed increased susceptibility to CSDS and impaired responses to ketamine treatment in the forced swim test. Floxed mice with conditional pan-neuronal (Synapsin-Cre) but not those with forebrain (αCaMKII-Cre) Vgf ablation displayed increased susceptibility to subthreshold social defeat stress, suggesting that neuronal VGF, expressed in part in inhibitory interneurons, regulates depression-like behavior. Acute antibody-mediated sequestration of VGF-derived C-terminal peptides AQEE-30 and TLQP-62 in dHc induced pro-depressant effects. Conversely, dHc TLQP-62 infusion had rapid antidepressant efficacy, which was reduced in BDNF floxed mice injected in dHc with AAV-Cre, and in NBQX- and rapamycin-pretreated wild-type mice, these compounds blocking α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and mammalian target of rapamycin (mTOR) signaling, respectively. VGF is therefore a critical modulator of depression-like behaviors in dHc and NAc. In hippocampus, the antidepressant response to ketamine is associated with rapid VGF translation, is impaired by reduced VGF expression, and as previously reported, requires coincident, rapid BDNF translation and release.

Altered peripheral immune profiles in treatment-resistant depression: response to ketamine and prediction of treatment outcome.

A subset of patients with depression have elevated levels of inflammatory cytokines, and some studies demonstrate interaction between inflammatory factors and treatment outcome. However, most studies focus on only a narrow subset of factors in a patient sample. In the current study, we analyzed broad immune profiles in blood from patients with treatment-resistant depression (TRD) at baseline and following treatment with the glutamate modulator ketamine. Serum was analyzed from 26 healthy control and 33 actively depressed TRD patients free of antidepressant medication, and matched for age, sex and body mass index. All subjects provided baseline blood samples, and TRD subjects had additional blood draw at 4 and 24 h following intravenous infusion of ketamine (0.5 mg kg-1). Samples underwent multiplex analysis of 41 cytokines, chemokines and growth factors using quantitative immunoassay technology. Our a priori hypothesis was that TRD patients would show elevations in canonical pro-inflammatory cytokines; analyses demonstrated significant elevation of the pro-inflammatory cytokine interleukin-6. Further exploratory analyses revealed significant regulation of four additional soluble factors in patients with TRD. Several cytokines showed transient changes in level after ketamine, but none correlated with treatment response. Low pretreatment levels of fibroblast growth factor 2 were associated with ketamine treatment response. In sum, we found that patients with TRD demonstrate a unique pattern of increased inflammatory mediators, chemokines and colony-stimulating factors, providing support for the immune hypothesis of TRD. These patterns suggest novel treatment targets for the subset of patients with TRD who evidence dysregulated immune functioning.

Persistent conditioned place preference to aggression experience in adult male sexually-experienced CD-1 mice.

We recently developed a conditioned place preference (CPP) procedure, commonly used to study rewarding drug effects, to demonstrate that dominant sexually-experienced CD-1 male mice form CPP to contexts previously associated with defeating subordinate male C57BL/6J mice. Here we further characterized conditioned and unconditioned aggression behavior in CD-1 mice. In Exp. 1 we used CD-1 mice that displayed a variable spectrum of unconditioned aggressive behavior toward younger subordinate C57BL/6J intruder mice. We then trained the CD-1 mice in the CPP procedure where one context was intruder-paired, while a different context was not. We then tested for aggression CPP 1 day after training. In Exp. 2, we tested CD-1 mice for aggression CPP 1 day and 18 days after training. In Exp. 3-4, we trained the CD-1 mice to lever-press for palatable food and tested them for footshock punishment-induced suppression of food-reinforced responding. In Exp. 5, we characterized unconditioned aggression in hybrid CD-1 × C57BL/6J D1-Cre or D2-Cre F1 generation crosses. Persistent aggression CPP was observed in CD-1 mice that either immediately attacked C57BL/6J mice during all screening sessions or mice that gradually developed aggressive behavior during the screening phase. In contrast, CD-1 mice that did not attack the C57BL/6J mice during screening did not develop CPP to contexts previously paired with C57BL/6J mice. The aggressive phenotype did not predict resistance to punishment-induced suppression of food-reinforced responding. CD-1 × D1-Cre or D2-Cre F1 transgenic mice showed strong unconditioned aggression. Our study demonstrates that aggression experience causes persistent CPP and introduces transgenic mice for circuit studies of aggression.

Pathogenesis of depression: Insights from human and rodent studies.

Major depressive disorder (MDD) will affect one out of every five people in their lifetime and is the leading cause of disability worldwide. Nevertheless, mechanisms associated with the pathogenesis of MDD have yet to be completely understood and current treatments remain ineffective in a large subset of patients. In this review, we summarize the most recent discoveries and insights for which parallel findings have been obtained in human depressed subjects and rodent models of mood disorders in order to examine the potential etiology of depression. These mechanisms range from synaptic plasticity mechanisms to epigenetics and the immune system where there is strong evidence to support a functional role in the development of specific depression symptomology. Ultimately we conclude by discussing how novel therapeutic strategies targeting central and peripheral processes might ultimately aid in the development of effective new treatments for MDD and related stress disorders.

Molecular mechanisms of psychostimulant-induced structural plasticity.

Drug addiction is characterized by persistent behavioral and cellular plasticity throughout the brain's reward regions. Among the many neuroadaptations that occur following repeated drug administration are alterations in cell morphology including changes in dendritic spines. While this phenomenon has been well documented, the underlying molecular mechanisms are poorly understood. Here, within the context of drug abuse, we review and integrate several of the established pathways known to regulate synaptic remodeling, and discuss the contributions of neurotrophic and dopamine signaling in mediating this structural plasticity. Finally, we discuss how such upstream mechanisms could regulate actin dynamics, the common endpoint involved in structural remodeling in neurons.

Phosphorylation of DeltaFosB mediates its stability in vivo.

The transcription factor, DeltaFosB, accumulates in a region-specific manner in brain in response to many types of chronic stimulation due to the unusual stability of the protein. The phosphorylation of Ser27 in DeltaFosB has been shown to promote this stability in vitro. We show here that this phosphorylation reaction is also important for DeltaFosB's stability in the brain in vivo and for the unique behavioral plasticity mediated by this transcription factor.

Gonadal hormones differentially modulate cocaine-induced conditioned place preference in male and female rats.

There is accumulating evidence that suggests there are sex differences in behavioral and subjective responses to cocaine. However, it is not known whether differences in cocaine reward contribute to sex differences in these responses or whether gonadal hormones affect the rewarding properties of cocaine. In the present study, conditioned place preference (CPP), a measure of non-contingent reward, was used to determine the effects of endogenous gonadal hormones and of estrogen and progesterone replacement on cocaine reward. Neurochemical measurements were also taken to identify monoaminergic substrates which underlie the behavioral phenotype. Although both intact and gonadectomized male and female rats showed a significant CPP for cocaine, ovariectomy attenuated the magnitude of CPP. These alterations coincided with a decrease in serum levels of corticosterone. In ovariectomized rats, pretreatment with progesterone inhibited cocaine CPP while estrogen plus progesterone potentiated the magnitude of CPP. Additionally, gonadectomy and ovarian hormone replacement in female rats affected serotonin/dopamine levels and turnover ratios in the ventral tegmental area and nucleus accumbens shell. While no effects of castration were observed, ovariectomy decreased levels of dopamine and serotonin in the ventral tegmental area. In females, progesterone replacement increased levels of serotonin and dopamine in the ventral tegmental area, while estrogen plus progesterone replacement increased dopamine levels in the nucleus accumbens. Collectively, these results indicate that ovarian hormones may influence cocaine reward by altering monoaminergic systems, which, in turn, may contribute to the current sex disparities in overall cocaine use.

Frequency of cocaine administration affects behavioral and endocrine responses in male and female Fischer rats.

Accumulating evidence has shown disparate behavioral responses to cocaine in male and female rats. To date, there is a lack of understanding of how cocaine administration frequency affects sexually dimorphic behavioral responses. In the present study we investigated the behavioral and endocrine responses to single (1 x 15 mg/kg) and "binge" (3 x 15 mg/kg) cocaine administration in male and female Fischer rats. Overall, females showed a more prolonged and robust behavioral response to both acute and "binge" pattern cocaine administration. Furthermore, sex-dependent behavioral topographies emerged during binge-pattern cocaine administration; female rearing activity increased across "binge" injections while ambulatory activity decreased. In contrast, male ambulatory and rearing behaviors remained constant across injections of "binge" cocaine. At the hormonal level, both single and "binge" pattern cocaine administration decreased testosterone levels in male rats. However, cocaine's modulation of testosterone levels was transient since testosterone levels were decreased by cocaine 30 min but not 3 hr following a single injection. In both male and female rats, "binge" cocaine increased plasma progesterone levels. However, acute cocaine administration increased progesterone levels transiently in only female rats. Our results show that pattern of administration affects both cocaine-stimulated behavioral and endocrine responses in male and female rats.

Temporal effects of estrogen and progesterone on behavioral and endocrinological responses to acute cocaine administration.

Estrogen and progesterone have been postulated to play a key role modulating cocaine-induced behavioral and neurochemical activation in female rats. This study investigated the temporal relationship between estrogen and progesterone in the modulation of cocaine-induced behavioral alterations. Ovariectomized Fischer rats received s.c. injections of estradiol benzoate 48 hr prior to cocaine or saline treatment and one s.c. injection of progesterone concurrently or 1, 4, 20, 24, 30, 44 or 48 hr after estrogen treatment. Forty-eight hours after estrogen treatment rats received either a single i.p. injection of 15 mg/kg of cocaine or 0.9% saline. Overall, cocaine induced increases in locomotor behaviors (ambulatory and rearing activity). A bimodal interaction between estrogen and progesterone was observed in the modulation of all locomotor activities. A gradual increase in behaviors, which peaked when progesterone was administered 24 hr after estrogen was followed by an inhibition of both ambulatory and rearing activity when progesterone was administered for a shorter period of time. This estrogen and progesterone interaction was not observed in the modulation of cocaine-induced stereotypic activity. However, shorter administration of progesterone in relation to estrogen administration resulted in lowered benzoylecgonine plasma levels when compared to longer progesterone administration times. On the other hand, longer administration of progesterone (48 hr of estrogen and progesterone) caused increases in corticosterone levels in cocaine-treated rats. Thus, the temporal interaction between estrogen and progesterone in the regulation of cocaine metabolism and hypothalamic-pituitary-axis (HPA) activation do not completely correlate with that observed for locomotor behavioral activation. Taken together, these results suggest that temporal interactions between estrogen and progesterone may underlie some of the previously reported estrous cycle and sex effects on cocaine-induced behavioral and endocrinological alteration.

The effect of low-Dye taping on peak plantar pressures of normal feet during gait.

This study investigated whether low-Dye anti-pronation taping altered peak plantar pressures of normal feet during gait. The Emed-AT-2 platform system was used to measure peak plantar pressures. Forty subjects performed two sets of six walks over the Emed-AT-2 forceplate. One set of walks was performed barefoot whilst the other set was performed with the low-Dye tape applied to the right foot. Computer software divided the heel, midfoot and forefoot into six areas (masks) for analysis. The mean for the peak plantar pressures (N/cm(2)) of each of these masks was determined for both sets of walks. Paired t-tests found a significant difference between the barefoot and taped peak plantar pressures in each of the six masks. Overall low-Dye anti-pronation taping significantly altered the peak plantar pressures of normal feet during gait. Of particular interest was that a significant reduction in mean peak plantar pressure was observed in the medial midfoot (1.4 N/cm(2)) whilst a significant increase occurred in the lateral midfoot (2.6 N/cm(2)).

Endocrinological basis of sex differences in cocaine-induced behavioral responses.

Currently, 1.8 million Americans use cocaine, 30% of whom are females. Sex differences in the pattern of cocaine abuse may reside in neuroendocrinological modulations that affect the use of and/or dependence on cocaine. This review discusses sex differences in cocaine-induced behavioral and molecular alterations in the central nervous system, with emphasis on the role of endocrine responses in the neuronal modulations of this drug. Mechanisms and data supporting the role of the hypothalamic-gonadal axis in the modulation of cocaine-induced behavioral and molecular alterations are also provided.

Ovarian hormones modulate cocaine-induced locomotor and stereotypic activity.

Interactions between ovarian hormones and cocaine may underlie gender and estrous cycle differences in cocaine-induced behavioral and neurochemical alterations. The aim of this study was to further understand how ovarian hormones modulate cocaine-induced behavioral alterations. Ovariectomized rats received acute or chronic saline or cocaine (15 mg/kg i.p.) administration and were further subdivided into one of four hormone-treatment conditions: cholesterol (vehicle-control), estrogen, progesterone, or estrogen + progesterone. Overall, acute and chronic cocaine administration increased all locomotor measurements (total locomotor, ambulatory, and rearing counts). Estrogen administration augmented cocaine-induced increases in ambulatory and rearing activity. After chronic cocaine administration, rats in the vehicle-control group developed behavioral tolerance (exhibited by a decrease in activity) in rearing and ambulatory activity. Estrogen replacement not only prevented the development of tolerance in ambulatory and rearing activities, but also enhanced total locomotor activity (sensitization) in response to chronic cocaine. Progesterone treatment did not alter the behavioral responses to acute or chronic cocaine administration. Estrogen + progesterone-treated animals had higher counts of locomotor activity in response to chronic cocaine than did vehicle-control or progesterone-treated rats. In stereotypic behaviors, the different hormonal treatments did not affect activity in cocaine- or saline-treated rats after acute or chronic drug administration. Plasma levels of cocaine did not change after different hormonal treatments. Interestingly, animals' coadministered chronic cocaine and estrogen had higher levels of corticosterone than did nonestrogen cocaine-treated rats. Thus, it is likely that alterations in HPA activation may underlie the observed behavioral differences. In summary, these data suggest that there are interactions between ovarian hormones and cocaine-induced behavioral alterations in female rats, and they extend previous results by showing that estrogen and progesterone affect the development of sensitization.

Post-translational processing of surfactant protein-C proprotein: targeting motifs in the NH(2)-terminal flanking domain are cleaved in late compartments.

Rat surfactant protein (SP)-C is a 3.7-kD hydrophobic lung-specific protein generated from proteolytic processing of a 21-kD propeptide (SP-C(21)). We have demonstrated that initial post-translational processing of SP-C(21) involves two cleavages of the COOH-terminus (Beers and colleagues, J. Biol. Chem. 1994;269:20,318--20,328). The goal of the current study was to define processing and function of the NH(2)-terminal flanking domain. Epitope-specific antisera directed against spatially distinct regions of the NH(2) terminus, NPROSP-C(2-9) (epitope = D(2)-L(9)) and NPROSP-C(11-23) (= E(11)-Q(23)) were produced. By Western blotting, both antisera identified SP-C(21) in microsomes. A 6-kD form (SP-C(6)), enriched in lamellar bodies (LBs), was detected only by NPROSP-C(11-23) and not extractable with NaCO(3) treatment. Immunogold staining of ultrathin lung sections with NPROSP-C(11-23) identified proSP-C in both multivesicular bodies (mvb) and LBs whereas NPROSP-C(2-9) labeled only mvb. (35)S-pulse chase analysis demonstrated synthesis of SP-C(21) and three intermediate forms (SP-C(16), SP-C(7), and SP-C(6)). Complete processing involved four separate cleavages with a precursor- product relationship between the low molecular weight forms SP-C(7) and SP-C(6). Fluorescence microscopy of A549 cells expressing fusion proteins of enhanced green fluorescent protein (EGFP) and proSP-C NH(2)-terminal deletion mutants showed targeting of EGFP/SP-C(1-194) and EGFP/SP-C(10-194) to early endosomal antigen-1-negative, CD-63-positive cytoplasmic vesicles whereas EGFP/SP-C(19-194), EGFP/SP-C(Delta 10-18), and EGFP/SP-C(24-194) were restricted to the endoplasmic reticulum (ER). We conclude that synthetic processing includes a previously unrecognized cleavage of the proximal NH(2) terminus (M(1)-L(9)), which occurs after removal of COOH-flanking domains (H(59)-I(194)) but before packaging in LBs, and that the region M(10)-T(18) is required for targeting of proSP-C to post-ER vesicular compartments in the biosynthetic pathway.

Vendor differences in cocaine-induced behavioral activity and hormonal interactions in ovariectomized Fischer rats.

Contradictory effects of ovarian hormone on cocaine-induced behaviors have been reported in ovariectomized Fischer rats. To determine if these discrepancies are based on where the rats were purchased, Charles River Laboratories and Taconic Fischer rats were randomly assigned to either cocaine (15 mg/kg, intraperitoneal) or saline treatment; and one of four hormone-pretreatment subgroups: vehicle, estrogen, progesterone or estrogen + progesterone. Vendor differences were observed in cocaine-induced locomotor activities; overall, Taconic rats demonstrated less locomotor activity than Charles River rats. Furthermore, vendor differences in ambulatory activity were also observed after steroid replacement treatment. In Charles River rats, estrogen + progesterone co-administration suppressed cocaine-induced increases in ambulatory activity when compared to other hormone-treated groups given cocaine. In contrast, Taconic rats showed an increase in ambulations after this drug/hormone treatment. Vendor differences were also observed in steroid effects on cocaine-induced rearing activity, where estrogen + progesterone and cocaine caused an increase in rearing in Charles River rats, but not in Taconic rats. No differences between the vendors were observed in saline- or cocaine-treated animals' stereotypic activity. Vendor differences in cocaine-induced locomotor activity were not due to differences in cocaine metabolism, as no differences in plasma levels of benzoylecgonine were observed. Interestingly, Taconic animals had overall higher plasma levels of corticosterone than Charles River rats. Thus, intrinsic differences between different lines of Fischer rats may affect the outcome of ovarian hormone interactions in cocaine-induced behavioral alterations.

Biosynthesis of surfactant protein C: characterization of aggresome formation by EGFP chimeras containing propeptide mutants lacking conserved cysteine residues.

Surfactant protein C (SP-C) is a lung-specific secreted protein, which is synthesized as a 21-kDa propeptide (SP-C(21)) and then proteolytically processed as a bitopic transmembrane protein in subcellular compartments distal to the medial Golgi to produce a 3.7 kDa mature form. We have shown that initial processing of SP-C(21) involves two endoproteolytic cleavages of the C terminus and that truncation of nine amino acids from the C-flanking peptide resulted in retention of mutant protein in proximal compartments. Because these truncations involved removal of a conserved cysteine residue (Cys(186)), we hypothesized that intralumenal disulfide-mediated folding of the C terminus of SP-C(21) is required for intracellular trafficking. To test this, cDNA constructs encoding heterologous fusion proteins consisting of enhanced green fluorescent protein (EGFP) attached to the N terminus of wild-type rat proSP-C (EGFP/SP-C(1-194)), C-terminally deleted proSP-C (EGFP/SP-C(1-185); EGFP/SP-C(1-191)) or point mutations of conserved cysteine residues (EGFP/SP-C(C122G); EGFP/SP-C(C186G); or EGFP/SP-C(C122/186G)) were transfected into A549 cells. Fluorescence microscopy revealed that transfected EGFP/SP-C(1-194) and EGFP/SP-C(1-191 )were expressed in a punctate pattern within CD-63 positive, EEA-1 negative cytoplasmic vesicles. In contrast, EGFP/SP-C(1-185), EGFP/SP-C(C122G), EGFP/SP-C(C186G) and EGFP/SP-C(C122/186G) were expressed but retained in a juxtanuclear compartment that stained for ubiquitin and that contained (&ggr;)-tubulin and vimentin, consistent with expression in aggresomes. Treatment of cells transfected with mutant proSP-C with the proteasome inhibitor lactacysteine enhanced aggresome formation, which could be blocked by coincubation with nocodazole. Western blots using a GFP antibody detected a single form in lysates of cells transfected with EGFP/SP-C cysteine mutants, without evidence of smaller degradation fragments. We conclude that residues Cys(122) and Cys(186) of proSP-C are required for proper post-translational trafficking. Mutation or deletion of one or both of these residues results in misfolding with mistargeting of unprocessed mutant protein, leading to formation of stable aggregates within aggresomes.

Structural requirements for intracellular targeting of SP-C proprotein.

Rat surfactant protein (SP) C is synthesized as a 194-amino acid proprotein that is proteolytically processed to a 35-amino acid mature form in subcellular compartments distal to the medial Golgi compartment. To identify domains of SP-C proprotein (proSP-C) necessary for endoplasmic reticulum translocation and for targeting to cytosolic processing compartments, we characterized expression patterns of heterologous SP-C fusion proteins in A549 lung epithelial cells and in the rat pheochromocytoma cell line PC-12. cDNA constructs were produced; these constructs encoded fusion proteins consisting of enhanced green fluorescent protein (EGFP) and wild-type proSP-C (EGFP/SP-C(1-194)), mature SP-C (EGFP/SP-C(24-59)), or progressive deletions of the NH(2)- or COOH-terminal flanking domains. By fluorescence microscopy, EGFP/SP-C(1-194) transfected into A549 cells was translocated and expressed in acidic cytoplasmic vesicles. By deletional analysis, a functional signal peptide was mapped to the domain Phe(24) to His(59), whereas a motif for targeting to cytosolic vesicular compartments was localized to the NH(2) flanking domain Met(10) to Gln(23). Truncations of the distal COOH terminus were retained in the endoplasmic reticulum/Golgi compartment; however, the COOH flanking region alone was insufficient for targeting. In PC-12 cells, EGFP/SP-C(1-194) was expressed in peripheral cytosolic vesicles, whereas EGFP/SP-C(24-194) and EGFP/SP-C(24-59) were each translocated but not targeted. We conclude that two domains in the proSP-C sequence are required for targeting: mature SP-C (Phe(24) to Leu(58)) contains a functional signal sequence active in epithelial and nonepithelial cells, whereas Met(10) to Gln(23), but not the COOH flanking peptide, is required for targeting to late vesicular compartments.

Synthetic processing of surfactant protein C by alevolar epithelial cells. The COOH terminus of proSP-C is required for post-translational targeting and proteolysis.

Surfactant protein C (SP-C) is synthesized by alveolar type II cells as a 21-kDa propeptide (proSP-C21) which is proteolytically processed in subcellular compartments distal to the trans-Golgi network to yield a 35-residue mature form. Initial synthetic processing events for SP-C include post-translational cleavages of the COOH terminus of proSP-C21 yielding two intermediates (16 and 6 kDa). To test the role of specific COOH-terminal domains in intracellular targeting and proteolysis of proSP-C21, synthesis and processing of SP-C was evaluated using a lung epithelial cell line (A549) transfected with a eukaryotic expression vector containing either the full-length cDNA for rat SP-C (SP-Cwt) or one of six polymerase chain reaction (PCR)-generated COOH terminally truncated forms (SP-C1-185, SP-C1-175, SP-C1-147, SP-C1-120, SP-C1-72, and SP-C1-59). Using in vitro transcription/translation, each of the seven constructs produced a 35S-labeled product of appropriate length which could be immunoprecipitated by epitope specific proSP-C antisera. Immunoprecipitation of 35S-labeled A549 cell lysates from SP-Cwt transfectants demonstrated rapid synthesis of [35S]proSP-C21 with processing to SP-C16 and SP-C6 intermediates via cleavages of the COOH-terminal propeptide. Both the intermediates as well as the kinetics of processing in A549 cells were similar to that observed in rat type II cells. In contrast, constructs SP-C1-185, SP-C1-175, SP-C1-147, SP-C1-120, SP-C1-72, and SP-C1-59 were each translated but degraded without evidence of proteolytic processing. Fluorescence immunocytochemistry identified proSP-Cwt in cytoplasmic vesicles of A549 cells while all COOH-terminal deletional mutants were restricted to an endoplasmic reticulum/Golgi compartment identified by co-localization with fluorescein isothiocyanate-concanavalin A. We conclude that SP-Cwt expressed in A549 cells is directed to cytoplasmic vesicles where it is proteolytically processed in a manner similar to native type II cells and that amino acids Cys186-Ile194 located at the COOH terminus of proSP-C21 are necessary for correct intracellular targeting and subsequent cleavage events.