Form of dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A nonphosphorylated at tyrosine 145 and 147 is enriched in the nuclei of astroglial cells, adult hippocampal progenitors, and some cholinergic axon terminals.

Compelling lines of evidence indicate that overexpression of dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) in subjects with trisomy 21 (Down syndrome[DS]) contributes to the abnormal structure and function of the DS brain. In the present study, we used a novel, phospho-dependent antibody recognizing DYRK1A only with nonphosphorylated tyrosine 145 and 147 (DYRK1A Tyr-145/147P(-)), to investigate the expression pattern of this DYRK1A species in trisomic and disomic human and mouse brains. Immunoblotting and dephosphorylation experiments demonstrated higher levels of DYRK1A Tyr-145/147P(-) in postnatal trisomic brains in comparison with controls (by ∼40%) than those of the DYRK1A visualized by three other N- and C-terminally directed antibodies to DYRK1A. By immunofluorescence, the immunoreactivity to DYRK1A Tyr-145/147P(-) was the strongest in the nuclei of astroglial cells, which contrasted with the predominantly neuronal localization of DYRK1A visualized by the three other antibodies to DYRK1A we used. In addition, DYRK1A Tyr-145/147P(-) was enriched in the nuclei of neuronal progenitors and newly born neurons in the adult hippocampal proliferative zone and also occurred in some cholinergic axonal terminals. Our data show a distinctive expression pattern of DYRK1A forms nonphosphorylated at Tyr-145 and Tyr-147 in the brain tissue and suggest that DS subjects may exhibit not only upregulation of total DYRK1A, but also more subtle differences in phosphorylation levels of this kinase in comparison with control individuals.

Amyloid-beta impairs development of neuronal progenitor cells by oxidative mechanisms.

Neuronal progenitor cells (NPCs) are being considered for treatment of neurodegenerative diseases associated with beta-amyloidosis: Alzheimer's disease (AD) and Down syndrome (DS). However, the neurotoxic properties of amyloid-beta peptide (Abeta) may impair survival and differentiation of transplanted NPCs. Hence, we studied the influence of Abeta on development of human NPCs--proliferation, migration, formation of colonies of neurons, formation processes--in culture. Pre-fibrillized human Abeta1-40 blocked development of neuronal colonies. NPC development was impaired in the presence of soluble Abeta1-40 (1.75-7 microM), and NPC differentiation into large and small neurons was altered, as demonstrated by morphometry. Antioxidant vitamin E partially abolished these effects, but not the reduced formation of neuronal processes. NPCs cultured with 7 microM Abeta1-40 accumulated Abeta monomers and oligomers and contained higher levels of protein carbonyls and lipid peroxidation products HNE and MDA. We suggest that Abeta1-40 impairs development of NPCs by oxidative damage. Hence, a prerequisite of successful neuroreplacement therapy using NPCs in AD and DS/AD may be removal of amyloid-beta and antioxidative treatment.

Tripeptidyl-peptidase I in neuronal ceroid lipofuscinoses and other lysosomal storage disorders.

The classic late infantile form of neuronal ceroid lipofuscinosis (CLN2, cLINCL) is associated with mutations in the gene encoding tripeptidyl-peptidase I (TPP-I), a lysosomal aminopeptidase that cleaves off tripeptides from the free N-termini of oligopeptides. To date over 30 different mutations and 14 polymorphisms associated with CLN2 disease process have been identified. In the present study, we analysed the molecular basis of 15 different mutations of TPP-I by using immunocytochemistry, immunofluorescence, Western blotting, enzymatic assay and subcellular fractionation. In addition, we studied the expression of TPP-I in other lysosomal storage disorders such as CLN1, CLN3, muccopolysaccharidoses and GM1 and GM2 gangliosidoses. Our study shows that TPP-I is absent or appears in very small amounts not only in cLINCL subjects with mutations producing severely truncated protein, but also in individuals with missense point mutations, which correlates with loss of TPP-I activity. Of interest, small amounts of TPP-I were detected in lysosomal fraction from fibroblasts from cLINCL subject with protracted form. This observation suggests that the presence of small amounts of TPP-I in lysosomes is able to delay significantly CLN2 disease process. We also show that TPP-I immunoreactivity is increased in the brain tissue of CLN1 and CLN3 subjects, stronger in glial cells and macrophages than neurons. Less prominent increase of TPP-I staining was found in muccopolysaccharidoses and GM1 and GM2 gangliosidoses. These data suggest that TPP-I participates in lysosomal turnover of proteins in pathological conditions associated with cell/tissue injury.

CLN3 disease process: missense point mutations and protein depletion in vitro.

Although the CLN3 gene associated with the disease process in subjects with the juvenile form of neuronal ceroid lipofuscinosis was discovered in 1995, our knowledge of the physiological function of its gene product, CLN3 protein, is still incomplete. To gain more insight into the structural properties and function of CLN3 protein we studied at present: i) how the naturally occurring point mutations Arg334Cys and Leu101Pro affect the biological properties of CLN3 protein, and ii) whether depletion of CLN3 protein synthesis by using an antisense approach induces a distinct phenotype in cells of neuronal origin in vitro. Here we report that although both CLN3 mutant proteins are targeted to lysosomes, thus similar to wild-type CLN3 protein, they are devoid of the biological activity of wild-type CLN3 protein such as its effect on lysosomal pH or intracellular processing of amyloid-beta protein precursor and cathepsin D in vitro. The Leu101Pro mutation affected significantly the maturation and stability of CLN3 protein. The Arg334Cys mutation influenced mildly the maturation and turnover of CLN3 protein, but at the same time abolished the function of CLN3 protein in vitro, which suggests that the Arg334 may constitute a part of the active site of CLN3 protein. In addition, we show that depletion of CLN3 protein synthesis in human neuroblastoma cells in vitro induces outgrowth of long cellular processes and formation of cellular aggregates and affects the viability of these cells. This finding suggests that CLN3 protein is implicated in biological processes associated with the differentiation of cells of neuronal origin.

Neuronal ceroid lipofuscinoses: classification and diagnosis.

The neuronal ceroid lipofuscinoses (NCLs) are neurodegenerative disorders characterized by accumulation of ceroid lipopigment in lysosomes in various tissues and organs. The childhood forms of the NCLs represent the most common neurogenetic disorders of childhood and are inherited in an autosomal-recessive mode. The adult form of NCL is rare and shows either an autosomal-recessive or autosomal dominant mode of inheritance. Currently, five genes associated with various childhood forms of NCLs, designated CLN1, CLN2, CLN3, CLN5, and CLN8, have been isolated and characterized. Two of these genes, CLN1 and CLN2, encode lysosomal enzymes: palmitoyl protein thioesterase 1 (PPT1) and tripetidyl peptidase 1 (TPP1), respectively. CLN3, CLN5, and CLN8 encode proteins of predicted transmembrane topology, whose function has not been characterized yet. Two other genes, CLN6 and CLN7, have been assigned recently to small chromosomal regions. Gene(s) associated with the adult form of NCLs (CLN4) are at present unknown. This study summarizes the current classification and new diagnostic criteria of NCLs based on clinicopathological, biochemical, and molecular genetic data. Material includes 159 probands with NCL (37 CLNI, 72 classical CLN2, 10 variant LINCL, and 40 CLN3) collected at the New York State Institute for Basic Research in Developmental Disabilities (IBR) as well as a comprehensive review of the literature. The results of our study indicate that although only biochemical and molecular genetic studies allow for definitive diagnosis, ultrastructural studies of the biopsy material are still very useful. Thus, although treatments for NCLs are not available at present, the diagnosis has become better defined.

Cellular pathology and pathogenic aspects of neuronal ceroid lipofuscinoses.

Lysosomal accumulation of autofluorescent, ceroid lipopigment material in various tissues and organs is a common feature of the neuronal ceroid lipofuscinoses (NCLs). However, recent clinicopathologic and genetic studies have evidenced that NCLs encompass a group of highly heterogeneous disorders. In five of the eight NCL variants distinguished at present, genes associated with the disease process have been isolated and characterized (CLN1, CLN2, CLN3, CLN5, CLN8). Only products of two of these genes, CLN 1 and CLN2, have structural and functional properties of lysosomal enzymes. Nevertheless, according to the nature of the material accumulated in the lysosomes, NCLs in humans as well as natural animal models of these disorders can be divided into two major groups: those characterized by the prominent storage of saposins A and D, and those showing the predominance of subunit c of mitochondrial ATP synthase accumulation. Thus, taking into account the chemical character of the major component of the storage material, NCLs can be classified currently as proteinoses. Of importance, although lysosomal storage material accumulates in NCL subjects in various organs, only brain tissue shows severe dysfunction and cell death, another common feature of the NCL disease process. However, the relation between the genetic defects associated with the NCL forms, the accumulation of storage material, and tissue damage is still unknown. This chapter introduces the reader to the complex pathogenesis of NCLs and summarizes our current knowledge of the potential consequences of the genetic defects of NCL-associated proteins on the biology of the cell.

Distribution of tripeptidyl peptidase I in human tissues under normal and pathological conditions.

Tripeptidyl peptidase I (TPP I) is a lysosomal exopeptidase that cleaves tripeptides from the free N-termini of oligopeptides. Mutations in this enzyme are associated with the classic late-infantile form of neuronal ceroid lipofuscinosis (CLN2), an autosomal recessive disorder leading to severe brain damage. To gain more insight into CLN2 pathogenesis and the role of TPP I in human tissues in general, we analyzed the temporal and spatial distribution of TPP I in the brain and its localization in internal organs under normal and pathological conditions. We report that TPP I immunoreactivity appears in neurons late in gestation and increases gradually in the postnatal period, matching significantly the final differentiation and maturation of neural tissue. Endothelial cells, choroid plexus, microglial cells, and ependyma showed TPP I immunostaining distinctly earlier than neurons. Acquisition of the adult pattern of TPP I distribution in the brain at around the age of 2 years correlates with the onset of clinical signs in CLN2 subjects. In adults, TPP I was found in all types of cells in the brain and internal organs we studied, although the intensity of TPP I labeling varied among several types of cells and showed a noticeable predilection for cells and/or organs associated with peptide hormone and neuropeptide production. In addition, TPP I immunoreactivity was increased in aging brain, neurodegenerative and lysosomal storage disorders, and some differentiated neoplasms and was reduced in ischemic/anoxic areas and undifferentiated tumors. These findings suggest that TPP I is involved in general protein turnover and that its expression may be controlled by various regulatory mechanisms, which highlights the importance of this enzyme for normal function of cells and organs in humans.

CLN3 protein regulates lysosomal pH and alters intracellular processing of Alzheimer's amyloid-beta protein precursor and cathepsin D in human cells.

Maintenance of the appropriate pH in the intracellular vacuolar compartments is essential for normal cell function. Here, we report that CLN3 protein, which is associated with the juvenile form of neuronal ceroid lipofuscinosis (JNCL), participates in lysosomal pH homeostasis in human cells. We show that CLN3 protein increases lysosomal pH in cultured human embryonal kidney cells, whereas inhibition of CLN3 protein synthesis by antisense approach acidifies lysosomal compartments. These changes in lysosomal pH are sufficient to exert a significant biological effect and modify intracellular processing of amyloid-beta protein precursor and cathepsin D, model proteins whose metabolism is influenced by the pH of acidic organelles. Mutant CLN3 protein (R334C) that is associated with the classical JNCL phenotype was devoid of biological activities of wild-type CLN3 protein. These data suggest that the pathogenesis of juvenile neuronal ceroid lipofuscinosis is associated with altered acidification of lysosomal compartments. Furthermore, our study indicates that CLN3 protein affects metabolism of proteins essential for cell functions, such as amyloid-beta protein precursor, implicated in Alzheimer's disease pathogenesis.

Sodium dodecyl sulfate-resistant complexes of Alzheimer's amyloid beta-peptide with the N-terminal, receptor binding domain of apolipoprotein E.

Immunocytochemical, biochemical, and molecular genetic studies indicate that apolipoprotein E (apoE) plays an important role in the process of amyloidogenesis-beta. However, there is still no clear translation of these data into the pathogenesis of amyloidosis-beta. Previous studies demonstrated sodium dodecyl sulfate (SDS)-resistant binding of apoE to the main component of Alzheimer's amyloid-A beta and modulation of A beta aggregation by apoE in vitro. To more closely characterize apoE-A beta interactions, we have studied the binding of thrombolytic fragments of apoE3 to A beta in vitro by using SDS-polyacrylamide gel electrophoresis and intrinsic fluorescence quenching. Here we demonstrate that SDS-resistant binding of A beta is mediated by the receptor-binding, N-terminal domain of apoE3. Under native conditions, both the N- and C-terminal domains of apoE3 bind A beta; however, the former does so with higher affinity. We propose that the modulation of A beta binding to the N-terminal domain of apoE is a potential therapeutic target for the treatment of amyloidosis-beta.

Receptor-dependent cell stress and amyloid accumulation in systemic amyloidosis.

Accumulation of fibrils composed of amyloid A in tissues resulting in displacement of normal structures and cellular dysfunction is the characteristic feature of systemic amyloidoses. Here we show that RAGE, a multiligand immunoglobulin superfamily cell surface molecule, is a receptor for the amyloidogenic form of serum amyloid A. Interactions between RAGE and amyloid A induced cellular perturbation. In a mouse model, amyloid A accumulation, evidence of cell stress and expression of RAGE were closely linked. Antagonizing RAGE suppressed cell stress and amyloid deposition in mouse spleens. These data indicate that RAGE is a potential target for inhibiting accumulation of amyloid A and for limiting cellular dysfunction induced by amyloid A.

Studies of membrane association of CLN3 protein.

The product of the CLN3 gene is a novel protein of unknown function. Simulations using amphiphacy algorithms have shown that structurally CLN3 may be another candidate for the family of membranous proteins. Signals controlling intracellular targeting of many membrane proteins are present as short sequences within their cytoplasmic domains. In fact, the sequence of CLN3 protein contains several such signaling sequences, which are conserved among mammals. First, at the N-terminus, potential N-myristoylation motif is present. Second, the C-terminal part of CLN3 protein contains both the dileucine motif, which is a potential lysosomal targeting signal, and the prenylation motif. There is scanty evidence of lysosomal and/or mitochondrial localization of CLN3 protein. However, the question of where the functional site of the cln3 protein exists in vivo remains unanswered. From theoretical calculations, we hypothesized that CLN3 should be an integral part of the membranous micro-environment. First, to test this hypothesis, we initiated detergent-partitioning experiments, localizing CLN3 predominantly in a pool of membranous protein. Further studies have shown that CLN3 protein integrates spontaneously with cellular membranes. Second, based on the prenylation results of CLN3 protein in vitro, we discussed the possible topological consequences of C-terminal fragment of CLN3 protein.

Analysis of intracellular distribution and trafficking of the CLN3 protein in fusion with the green fluorescent protein in vitro.

CLN3 gene, associated with juvenile neuronal ceroid lipofuscinosis, encodes a novel protein of a predicted 438 amino acid residues. We have expressed a full-length CLN3 protein and fragments thereof in fusion with green fluorescent protein in Chinese hamster ovary and human neuroblastoma cell lines to study its subcellular localization and intracellular trafficking pattern. By using laser scanning confocal microscopy, we demonstrate that the full-length CLN3 fusion protein is targeted to lysosomal compartments. Tunicamycin treatment did not alter the lysosomal targeting of the CLN3 protein, which indicates that extensive N-glycosylation of the full-length CLN3 fusion protein is not engaged in its lysosomal sorting. Monensin produced retention of CLN3 fusion protein in vesicular structure of the Golgi apparatus in the perinuclear space, suggesting that CLN3 fusion protein is transported to the lysosomal compartments through the trans-Golgi cisternae. Neither of the truncated CLN3 fusion proteins encompassing its 1-138, 1-322, and 138-438 amino acid residues was disclosed in lysosomal compartments. However, CLN3 fusion protein showing double-point mutations at amino acid residues 425 and 426, thus at its putative dileucine lysosomal signaling motif, was still targeted to lysosomes, suggesting that a dileucine motif alone is not sufficient for lysosomal sorting of the CLN3 fusion protein.

Posttranslational modification of CLN3 protein and its possible functional implication.

The CLN3 gene associated with Batten disease and encoding a novel protein of a predicted 438 amino acids was cloned in 1995 by the International Batten Disease Consortium. The function of CLN3 protein remains unknown. Computer-based analysis predicted that CLN3 may contain several posttranslational modifications. Thus, to study the posttranslational modification of CLN3 protein, we have expressed a full-length CLN3 protein as a C-terminal fusion with green fluorescent protein of the jellyfish Aequerea victoria in a Chinese hamster ovary cell line. Previously, we have shown that CLN3 is a glycosylated protein from lysosomal compartment, and now, by using in vivo labeling with 32P, detection with anti-phosphoamino acid antibodies, and phosphoamino acid analysis, we demonstrate that CLN3 is a phosphorylated protein. We demonstrate that CLN3 protein does not undergo mannose 6-phosphate modification and that it is a membrane protein. Furthermore, we show that the level of CLN3 protein phosphorylation may be modulated by several protein kinases and phosphatases activators or inhibitors.

Expression studies of CLN3 protein (battenin) in fusion with the green fluorescent protein in mammalian cells in vitro.

The gene for Batten disease, the CLN3 gene, encodes a novel, highly hydrophobic, multitransmembrane protein, predicted to consist of 438 amino acid residues. We have expressed a full-length CLN3 protein in fusion with green fluorescent protein in various cell lines to provide its initial biochemical characterization and subcellular localization. By using Western blotting, Percoll density gradient fractionation, and Triton X-114 extraction, we demonstrate that the product of the CLN3 gene, which we call battenin, in mammalian expression system studied is a highly glycosylated protein of lysosomal membrane. In addition our data suggest that CLN3 protein is processed proteolytically in acidic compartments of the cell. Thus, battenin represents the novel constituent of a growing family of lysosomal membrane proteins.

Evidence for phosphorylation of CLN3 protein associated with Batten disease.

Recently, the CLN3 gene associated with Batten disease (juvenile neuronal ceroid lipofuscinosis, JNCL), a recessively inherited, progressive, neurodegenerative disorder of childhood, has been identified. The CLN3 gene encodes a novel protein (battenin) of a predicted 438 amino acids containing several potential posttranslational modifications. We have expressed a full-length CLN3 protein as a C-terminal fusion with green fluorescent protein (GFP) to evaluate whether CLN3 protein is phosphorylated. By using in vivo labeling with 32P, detection with anti-phosphoamino acid antibodies, and phosphoamino acid analysis, we demonstrate that the CLN3 protein is phosphorylated on both serine and threonine residues. We also demonstrate that CLN3 protein is not modified by mannose 6-phosphate. Furthermore, we show that phosphorylation of CLN3 protein is carried out by protein kinase A (cAMP-dependent protein kinase, PKA), protein kinase G (cGMP-dependent protein kinase, PKG), and casein kinase II and that it is enhanced by inhibition of protein phosphatase 1 (PP 1) or protein phosphatase 2A (PP 2A).

The "nonamyloidogenic" p3 fragment (amyloid beta17-42) is a major constituent of Down's syndrome cerebellar preamyloid.

Down's syndrome (DS) patients show accelerated Alzheimer's disease (AD) neuropathology, which consists of preamyloid lesions followed by the development of neuritic plaques and neurofibrillary tangles. The major constituents of preamyloid and neuritic plaques are amyloid beta (Abeta) peptides. Preamyloid lesions are defined as being Abeta immunoreactive lesions, which unlike neuritic plaque amyloid are Congo red-negative and largely nonfibrillar ultrastructurally. DS patients can develop extensive preamyloid deposits in the cerebellum, without neuritic plaques; hence, DS cerebellums are a source of relatively pure preamyloid. We biochemically characterized the composition of DS preamyloid and compared it to amyloid in the neuritic plaques and leptomeninges in the same patients. We found that Abeta17-42 or p3 is a major Abeta peptide of DS cerebellar preamyloid. This 26-residue peptide is also present in low quantities in neuritic plaques. We suggest that preamyloid can now be defined biochemically as lesions in which a major Abeta peptide is p3.

Micropreparative gel electrophoresis of low-molecular-weight peptides: purification of highly insoluble amyloid peptide fragments.

We have used the continuous-elution micropreparative gel electrophoresis device described by Baumann and Lauraeus (Anal. Biochem. 214, 142-148, 1993) to purify low-molecular-weight peptide fragments from in-gel digested standard proteins as well as highly in-soluble amyloid peptides. Alzheimer's amyloid beta-peptide, gelsolin-derived amyloid peptide of the Finnish type, and a novel amyloid of the British type were purified from either homogenized brain or kidney tissue material to a high degree of purity in a single run. Using the high resolving capacity of the Tris-Tricine-SDS buffer system of Schaegger and von Jagow (Anal. Biochem. 166, 368-379, 1978) we were able to isolate two synthetic peptides with M(r)4329 and 3284, differing only by 1045 in mass. The total peptide recovery, as determined by amino acid sequence analysis and scanning densitometry, ranged between 60 and 80%. In order to demonstrate the utility of this technique we subjected some of the purified peptides to direct N-terminal amino acid sequence analysis, mass spectrometry, microbore high-performance liquid chromatography, and immunochemical studies. Our results show that micropreparative gel electrophoresis is an effective tool for the isolation of not only larger polypeptides but also small peptide fragments in a form suitable for further biological use.

The interaction between apolipoprotein E and Alzheimer's amyloid beta-peptide is dependent on beta-peptide conformation.

An important feature of Alzheimer's disease (AD) is the cerebral deposition of amyloid. The main component of the amyloid is a 39-44-amino acid residue protein called amyloid beta (A beta), which also exists as a normal protein in biological fluids, known as soluble A beta. A major risk factor for late-onset AD is the inheritance of the apolipoprotein (apo) E4 isotype of apoE. How apoE is involved in the pathogenesis of AD is unclear; however, evidence exists for a direct apoE/A beta interaction. We and others have shown that apoE copurifies with A beta from AD amyloid plaques and that under certain in vitro conditions apoE promotes a beta-sheet structure in A beta peptides. Currently we document the high affinity binding of A beta peptides to both human recombinant apoE3 and -E4 with a KD of 20 nM. This interaction is greatly influenced by the conformational state of the A beta peptide used. Furthermore, we show that the fibril modulating effect of apoE is also influenced by the initial secondary structure of the A beta peptide. The preferential binding of apoE to A beta peptides with a beta-sheet conformation can in part explain the copurification of A beta and apoE from AD amyloid plaques.

Alzheimer's beta-amyloid peptide is conformationally modified by apolipoprotein E in vitro.

Amyloid beta-peptide (A beta) is a major component of neuritic plaques, a feature of Alzheimer's disease (AD) brains. Recently, we showed that A beta adopts two major conformational states in solution, which differ in their abilities to form amyloid. These are highly amyloidogenic conformer (A beta ac) with a high content of beta-sheet and a slowly amyloidogenic conformer (A beta nac) with a random coil conformation. Apolipoprotein E (apoE), particularly the E4 isoform, which is genetically associated with AD, binds to A beta and modulates fibrillogenesis in vitro. In the present work, the influence of apoE on the conformation of A beta peptides was studied. The results suggest that, under the conditions used, apoE enhances amyloid formation by inducing the conformational transition from A beta nac into A beta ac. We propose that an important step in A beta fibrillogenesis is the transformation induced by apoE of the soluble non-amyloidogenic into the pathological amyloidogenic conformer of A beta.