Long-term voluntary running modifies the levels of proteins of the excitatory/inhibitory system and reduces reactive astrogliosis in the brain of Ts65Dn mouse model for Down syndrome.

We showed previously that voluntary long-term running improved cognition and motor skills, but in an age-dependent manner, in the Ts65Dn mouse model for Down syndrome (DS). Presently, we investigated the effect of running on the levels of some key proteins of the excitatory/inhibitory system, which is impaired in the trisomic brain, and on astroglia, a vital component of this system. Ts65Dn mice had free access to a running wheel for 9-13 months either from weaning or from the age of 7 months. Sedentary Ts65Dn mice served as controls. We found that running modified the levels of four of the seven proteins we tested that are associated with the glutamatergic/GABA-ergic system. Thus, Ts65Dn runners demonstrated increased levels of glutamine synthetase and metabotropic glutamate receptor 1 and decreased levels of glutamate transporter 1 and glutamic acid decarboxylase 65 (GAD65) versus sedentary mice, but of metabotropic glutamate receptor 1 and GAD65 only in the post-weaning cohort. GAD67, ionotropic N-methyl-D-aspartate type receptor subunit 1, and GABAAα5 receptors' levels were similar in runners and sedentary animals. The number of glial fibrillary acidic protein (GFAP)-positive astrocytes and the levels of GFAP were significantly reduced in runners relative to sedentary mice. Our study provides new insight into the mechanisms underlying the beneficial effect of voluntary, sustained running on function of the trisomic brain by identifying the involvement of proteins associated with glutamatergic and GABAergic systems and reduction in reactive astrogliosis.

Increased levels of carbonic anhydrase II in the developing Down syndrome brain.

By using a proteomic approach, we found increased levels of carbonic anhydrase II (CA II) in the brain of Ts65Dn mice, a mouse model for Down syndrome (DS). Further immunoblot analyses showed that the levels of CA II are increased not only in the brain of adult Ts65Dn mice but also in the brain of infants and young children with DS. Cellular localization of the enzyme in human brain, predominantly in the oligodendroglia and primitive vessels in fetal brain and in the oligodendroglia and some GABAergic neurons postnatally, was similar in DS subjects and controls. Given the role of CA II in regulation of electrolyte and water balance and pH homeostasis, up-regulation of CA II may reflect a compensatory mechanism mobilized in response to structural/functional abnormalities in the developing DS brain. However, this up-regulation may also have an unfavorable effect by increasing susceptibility to seizures of children with DS.

Widespread cerebellar transcriptome changes in Ts65Dn Down syndrome mouse model after lifelong running.

Our previous study showed an improvement in locomotor deficits after voluntary lifelong running in Ts65Dn mice, an animal model for Down syndrome (DS). In the present study, we employed mouse microarrays printed with 55,681 probes in an attempt to identify molecular changes in the cerebellar transcriptome that might contribute to the observed behavioral benefits of voluntary long-term running in Ts65Dn mice. Euploid mice were processed in parallel for comparative purposes in some analyses. We found that running significantly changed the expression of 4,315 genes in the cerebellum of Ts65Dn mice, over five times more than in euploid animals, up-regulating 1,991 and down-regulating 2,324 genes. Functional analysis of these genes revealed a significant enrichment of 92 terms in the biological process category, including regulation of biosynthesis and metabolism, protein modification, phosphate metabolism, synaptic transmission, development, regulation of cell death/apoptosis, protein transport, development, neurogenesis and neuron differentiation. The KEGG pathway database identified 18 pathways that are up-regulated and two that are down-regulated by running that were associated with learning, memory, cell signaling, proteolysis, regeneration, cell cycle, proliferation, growth, migration, and survival. Of six mRNA protein products we tested by immunoblotting, four showed significant running-associated changes in their levels, the most prominent in glutaminergic receptor metabotropic 1, and two showed changes that were close to significant. Thus, unexpectedly, our data point to the high molecular plasticity of Ts65Dn mouse cerebellum, which translated into humans with DS, suggests that the motor deficits of individuals with DS could markedly benefit from prolonged exercise.

Long-term running alleviates some behavioral and molecular abnormalities in Down syndrome mouse model Ts65Dn.

Running may affect the mood, behavior and neurochemistry of running animals. In the present study, we investigated whether voluntary daily running, sustained over several months, might improve cognition and motor function and modify the brain levels of selected proteins (SOD1, DYRK1A, MAP2, APP and synaptophysin) in Ts65Dn mice, a mouse model for Down syndrome (DS). Ts65Dn and age-matched wild-type mice, all females, had free access to a running wheel either from the time of weaning (post-weaning cohort) or from around 7 months of age (adult cohort). Sedentary female mice were housed in similar cages, without running wheels. Behavioral testing and evaluation of motor performance showed that running improved cognitive function and motor skills in Ts65Dn mice. However, while a dramatic improvement in the locomotor functions and learning of motor skills was observed in Ts65Dn mice from both post-weaning and adult cohorts, improved object memory was seen only in Ts65Dn mice that had free access to the wheel from weaning. The total levels of APP and MAP2ab were reduced and the levels of SOD1 were increased in the runners from the post-weaning cohort, while only the levels of MAP2ab and α-cleaved C-terminal fragments of APP were reduced in the adult group in comparison with sedentary trisomic mice. Hence, our study demonstrates that Ts65Dn females benefit from sustained voluntary physical exercise, more prominently if running starts early in life, providing further support to the idea that a properly designed physical exercise program could be a valuable adjuvant to future pharmacotherapy for DS.

Molecular chaperone alphaB-crystallin is expressed in the human fetal telencephalon at midgestation by a subset of progenitor cells.

Alphab-crystallin (CRYAB) is a small heat shock protein with a chaperoning activity that is present in the postnatal healthy human brain in oligodendrocytes and in a few astrocytes. The involvement of CRYAB in cell differentiation, proliferation, signaling, cytoskeletal assembly, and apoptosis in various model systems has suggested that it might also play a role in the developing human brain. We analyzed the distribution and the levels of this molecular chaperone in healthy and polygenetically compromised (Down syndrome [DS]) human telencephalon at midgestation. We demonstrate that CRYAB is expressed in a temporospatial pattern by numerous radial glial cells and some early oligodendrocyte progenitors, including dividing cells, as well as a few astroglial cells in both healthy and DS fetal brains. We also found abundant phosphorylation of CRYAB at Ser-59, which mediates its antiapoptotic and cytoskeletal functions. There was only marginal phosphorylation at Ser-45.In contrast to our earlier study in young DS subjects, upregulation of phosphorylated CRYAB occurred rarely in DS fetuses. The distribution, the timing of appearance, and the results of colocalization studies suggest that CRYAB assists in the biological processes associated with developmental remodeling/differentiation and proliferation of select subpopulations of progenitor cells in human fetal brain at midgestation.

A critical tryptophan and Ca2+ in activation and catalysis of TPPI, the enzyme deficient in classic late-infantile neuronal ceroid lipofuscinosis.

BACKGROUND: Tripeptidyl aminopeptidase I (TPPI) is a crucial lysosomal enzyme that is deficient in the fatal neurodegenerative disorder called classic late-infantile neuronal ceroid lipofuscinosis (LINCL). It is involved in the catabolism of proteins in the lysosomes. Recent X-ray crystallographic studies have provided insights into the structural/functional aspects of TPPI catalysis, and indicated presence of an octahedrally coordinated Ca(2+). METHODOLOGY: Purified precursor and mature TPPI were used to study inhibition by NBS and EDTA using biochemical and immunological approaches. Site-directed mutagenesis with confocal imaging technique identified a critical W residue in TPPI activity, and the processing of precursor into mature enzyme. PRINCIPAL FINDINGS: NBS is a potent inhibitor of the purified TPPI. In mammalian TPPI, W542 is critical for tripeptidyl peptidase activity as well as autocatalysis. Transfection studies have indicated that mutants of the TPPI that harbor residues other than W at position 542 have delayed processing, and are retained in the ER rather than transported to lysosomes. EDTA inhibits the autocatalytic processing of the precursor TPPI. CONCLUSIONS/SIGNIFICANCE: We propose that W542 and Ca(2+) are critical for maintaining the proper tertiary structure of the precursor proprotein as well as the mature TPPI. Additionally, Ca(2+) is necessary for the autocatalytic processing of the precursor protein into the mature TPPI. We have identified NBS as a potent TPPI inhibitor, which led in delineating a critical role for W542 residue. Studies with such compounds will prove valuable in identifying the critical residues in the TPPI catalysis and its structure-function analysis.

Upregulation of phosphorylated alphaB-crystallin in the brain of children and young adults with Down syndrome.

Our previous proteomic studies disclosed upregulation of alphaB-crystallin, a small heat shock protein, in the brain tissue of Ts65Dn mice, a mouse model for Down syndrome (DS). To validate data obtained in model animals, we studied at present the levels and distribution of total alphaB-crystallin and its forms phosphorylated at Ser-45 and Ser-59 in the brain tissues of DS subjects and age-matched controls at 4 months to 23 years of age. On immunoblots from frontal cortex and white matter, alphaB-crystallin and its form phosphorylated at Ser-59 were detectable already in infants, whereas alphaB-crystallin phosphorylated at Ser-45 appeared in small amounts in older children. Although the levels of total alphaB-crystallin were modestly increased in DS subjects, the amounts of both phosphorylated forms were much higher (up to approximately 550%) in the group of older children and young adults with DS than in age-matched controls. Immunoreactivity to alphaB-crystallin occurred not only in a subset of oligodendrocytes and some subpial and perivascular astrocytes, which was reported earlier, but also in GFAP-positive astrocytes accumulating at the sites of ependymal injury as well as some GFAP/platelet-derived growth factor receptor alpha-positive cells in both DS and control brains, which is a novel observation. Given that the chaperone and anti-apoptotic activities of alphaB-crystallin are phosphorylation-dependent, we propose that enhanced phosphorylation of alphaB-crystallin in the brains of young DS subjects might reflect a cytoprotective mechanism mobilized in response to stress conditions induced or augmented by the effect of genes encoded by the triplicated chromosome 21.

Prosegment of tripeptidyl peptidase I is a potent, slow-binding inhibitor of its cognate enzyme.

Tripeptidyl peptidase I (TPP I) is the first mammalian representative of a family of pepstatin-insensitive serine-carboxyl proteases, or sedolisins. The enzyme acts in lysosomes, where it sequentially removes tripeptides from the unmodified N terminus of small, unstructured polypeptides. Naturally occurring mutations in TPP I underlie a neurodegenerative disorder of childhood, classic late infantile neuronal ceroid lipofuscinosis (CLN2). Generation of mature TPP I is associated with removal of a long prosegment of 176 amino acid residues from the zymogen. Here we investigated the inhibitory properties of TPP I prosegment expressed and isolated from Escherichia coli toward its cognate protease. We show that the TPP I prosegment is a potent, slow-binding inhibitor of its parent enzyme, with an overall inhibition constant in the low nanomolar range. We also demonstrate the protective effect of the prosegment on alkaline pH-induced inactivation of the enzyme. Interestingly, the inhibitory properties of TPP I prosegment with the introduced classic late infantile neuronal ceroid lipofuscinosis disease-associated mutation, G77R, significantly differed from those revealed by wild-type prosegment in both the mechanism of interaction and the inhibitory rate. This is the first characterization of the inhibitory action of the sedolisin prosegment.

Glycosaminoglycans modulate activation, activity, and stability of tripeptidyl-peptidase I in vitro and in vivo.

Tripeptidyl-peptidase I (TPP I, CLN2 protein) is a lysosomal exopeptidase that sequentially removes tripeptides from the N termini of polypeptides and shows a minor endoprotease activity. Mutations in TPP I lead to classic late-infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disease. TPP I proenzyme is converted in lysosomes into a mature enzyme with the assistance of another protease and is able to autoactivate in acidic pH in vitro via a unimolecular mechanism. Because autoactivation in vitro at the pH values reported for lysosomes generated inactive enzyme, we intended to determine whether physiologically relevant factors can modify this process to also make it plausible in vivo. Here, we report that high ionic strength and glycosaminoglycans (GAGs) increase yields (ionic strength) or yields and rates (GAGs) of activation, enhance degradation of liberated TPP I prosegment fragments, and switch effective autoactivation of TPP I proenzyme toward less acidic pH values (up to pH 6.0). Although ionic strength and GAGs also inhibited TPP I activity in vitro and in living cells, the degree of inhibition (from 20 to 60%) appears to be of rather limited functional significance. Importantly, binding to GAGs improved thermal stability of TPP I and protected the enzyme against alkaline pH-induced denaturation in vitro (t((1/2)) of mature enzyme at pH 7.4 increased by approximately 8-fold in the presence of heparin) and in vivo ( approximately 2-fold higher loss of TPP I in cells deficient in GAGs than in control cells after bafilomycin A1 treatment). These findings elucidate a potent physiologically relevant mechanism of TPP I regulation by GAGs and suggest that generation of the active enzyme via autoactivation can be accomplished not only in vitro but in vivo as well.

N-glycosylation is crucial for folding, trafficking, and stability of human tripeptidyl-peptidase I.

Tripeptidyl-peptidase I (TPP I) is a lysosomal serine-carboxyl peptidase that sequentially removes tripeptides from polypeptides. Naturally occurring mutations in TPP I are associated with the classic late infantile neuronal ceroid lipofuscinosis. Human TPP I has five potential N-glycosylation sites at Asn residues 210, 222, 286, 313, and 443. To analyze the role of N-glycosylation in the function of the enzyme, we obliterated each N- glycosylation consensus sequence by substituting Gln for Asn, either individually or in combinations, and expressed mutated cDNAs in Chinese hamster ovary and human embryonic kidney 293 cells. Here, we demonstrate that human TPP I in vivo utilizes all five N-glycosylation sites. Elimination of one of these sites, at Asn-286, dramatically affected the folding of the enzyme. However, in contrast to other misfolded proteins that are retained in the endoplasmic reticulum, only a fraction of misfolded TPP I mutant expressed in Chinese hamster ovary cells, but not in human embryonic kidney 293 cells, was arrested in the ER, whereas its major portion was secreted. Secreted proenzyme formed non-native, interchain disulfide bridges and displayed only residual TPP I activity upon acidification. A small portion of TPP I missing Asn-286-linked glycan reached the lysosome and was processed to an active species; however, it showed low thermal and pH stability. N-Glycans at Asn-210, Asn-222, Asn-313, and Asn-443 contributed slightly to the specific activity of the enzyme and its resistance to alkaline pH-induced inactivation. Phospholabeling experiments revealed that N-glycans at Asn-210 and Asn-286 of TPP I preferentially accept a phosphomannose marker. Thus, a dual role of oligosaccharide at Asn-286 in folding and lysosomal targeting could contribute to the unusual, but cell type-dependent, fate of misfolded TPP I conformer and represent the molecular basis of the disease process in subjects with naturally occurring missense mutation at Asn-286.

Biosynthesis, glycosylation, and enzymatic processing in vivo of human tripeptidyl-peptidase I.

Human tripeptidyl-peptidase I (TPP I, CLN2 protein) is a lysosomal serine protease that removes tripeptides from the free N termini of small polypeptides and also shows a minor endoprotease activity. Due to various naturally occurring mutations, an inherited deficiency of TPP I activity causes a fatal lysosomal storage disorder, classic late infantile neuronal ceroid lipofuscinosis (CLN2). In the present study, we analyzed biosynthesis, glycosylation, transport, and proteolytic processing of this enzyme in stably transfected Chinese hamster ovary cells as well as maturation of the endocytosed proenzyme in CLN2 lymphoblasts, fibroblasts, and N2a cells. Human TPP I was initially identified as a single precursor polypeptide of approximately 68 kDa, which, within a few hours, was converted to the mature enzyme of approximately 48 kDa. Compounds affecting the pH of intracellular acidic compartments, those interfering with the intracellular vesicular transport as well as inhibition of the fusion between late endosomes and lysosomes by temperature block or 3-methyladenine, hampered the conversion of TPP I proenzyme into the mature form, suggesting that this process takes place in lysosomal compartments. Digestion of immunoprecipitated TPP I proenzyme with both N-glycosidase F and endoglycosidase H as well as treatment of the cells with tunicamycin reduced the molecular mass of TPP I proenzyme by approximately 10 kDa, which indicates that all five potential N-glycosylation sites in TPP I are utilized. Mature TPP I was found to be partially resistant to endo H treatment; thus, some of its N-linked oligosaccharides are of the complex/hybrid type. Analysis of the effect of various classes of protease inhibitors and mutation of the active site Ser(475) on human TPP I maturation in cultured cells demonstrated that although TPP I zymogen is capable of autoactivation in vitro, a serine protease that is sensitive to AEBSF participates in processing of the proenzyme to the mature, active form in vivo.

Maturation of human tripeptidyl-peptidase I in vitro.

Tripeptidyl-peptidase I (TPP I, CLN2 protein) is a lysosomal aminopeptidase that cleaves off tripeptides from the free N termini of oligopeptides and also shows minor endopeptidase activity. TPP I is synthesized as a preproenzyme. Its proenzyme autoactivates under acidic conditions in vitro, resulting in a rapid conversion into the mature form. In this study, we examined the process of maturation in vitro of recombinant latent human TPP I purified to homogeneity from secretions of Chinese hamster ovary cells overexpressing TPP I cDNA. Autoprocessing of TPP I proenzyme was carried out at a wide pH range, from approximately 2.0 to 6.0, albeit with different efficiencies depending on the pH and the type of buffer. However, the acquisition of enzymatic activity in the same buffer took place in a narrower pH "window," usually in the range of 3.6-4.2. N-terminal sequencing revealed that mature, inactive enzyme generated during autoactivation at higher pH contained N-terminal extensions (starting at 6 and 14 amino acid residues upstream of the prosegment/mature enzyme junction), which could contribute to the lack of activity of TPP I generated in this manner. Autoprocessing was not associated with any major changes of the secondary structure of the proenzyme, as revealed by CD spectroscopy. Both the activation and proteolytic processing of the recombinant TPP I precursor were primarily concentration-independent. The addition of the mature enzyme did not accelerate the processing of the proenzyme. In addition, the maturation of the proenzyme was not affected by the presence of glycerol. Finally, the proenzyme with the active site mutated (S475L) was not processed in the presence of the wild-type enzyme. All of these findings indicate a primarily intramolecular (unimolecular) mechanism of TPP I activation and autoprocessing and suggest that in vivo mature enzyme does not significantly participate in its own generation from the precursor.