Untargeted Metabolite Profiling of Cerebrospinal Fluid Uncovers Biomarkers for Severity of Late Infantile Neuronal Ceroid Lipofuscinosis (CLN2, Batten Disease).

Late infantile neuronal ceroid lipofuscinosis (CLN2 disease) is a rare lysosomal storage disorder caused by a monogenetic deficiency of tripeptidyl peptidase-1 (TPP1). Despite knowledge that lipofuscin is the hallmark disease product, the relevant TPP1 substrate and its role in neuronal physiology/pathology is unknown. We hypothesized that untargeted metabolite profiling of cerebrospinal fluid (CSF) could be used as an effective tool to identify disease-associated metabolic disruptions in CLN2 disease, offering the potential to identify biomarkers that inform on disease severity and progression. Accordingly, a mass spectrometry-based untargeted metabolite profiling approach was employed to differentiate CSF from normal vs. CLN2 deficient individuals. Of 1,433 metabolite features surveyed, 29 linearly correlated with currently employed disease severity scores. With tandem mass spectrometry 8 distinct metabolite identities were structurally confirmed based on retention time and fragmentation pattern matches, vs. standards. These putative CLN2 biomarkers include 7 acetylated species - all attenuated in CLN2 compared to controls. Because acetate is the major bioenergetic fuel for support of mitochondrial respiration, deficient acetylated species in CSF suggests a brain energy defect that may drive neurodegeneration. Targeted analysis of these metabolites in CSF of CLN2 patients offers a powerful new approach for monitoring CLN2 disease progression and response to therapy.

AAV gene transfer delays disease onset in a TPP1-deficient canine model of the late infantile form of Batten disease.

The most common form of the childhood neurodegenerative disease late infantile neuronal ceroid lipofuscinosis (also called Batten disease) is caused by deficiency of the soluble lysosomal enzyme tripeptidyl peptidase 1 (TPP1) resulting from mutations in the TPP1 gene. We tested whether TPP1 gene transfer to the ependyma, the epithelial lining of the brain ventricular system, in TPP1-deficient dogs would be therapeutically beneficial. A one-time administration of recombinant adeno-associated virus (rAAV) expressing canine TPP1 (rAAV.caTPP1) resulted in high expression of TPP1 predominantly in ependymal cells and secretion of the enzyme into the cerebrospinal fluid leading to clinical benefit. Diseased dogs treated with rAAV.caTPP1 showed delays in onset of clinical signs and disease progression, protection from cognitive decline, and extension of life span. By immunostaining and enzyme assay, recombinant protein was evident throughout the brain and spinal cord, with correction of the neuropathology characteristic of the disease. This study in a naturally occurring canine model of TPP1 deficiency highlights the utility of AAV transduction of ventricular lining cells to accomplish stable secretion of recombinant protein for broad distribution in the central nervous system and therapeutic benefit.

Cerebrospinal fluid proteomic analysis reveals dysregulation of methionine aminopeptidase-2 expression in human and mouse neurofibromatosis 1-associated glioma.

Individuals affected with the neurofibromatosis 1 (NF1) tumor predisposition syndrome are prone to the development of multiple nervous system tumors, including optic pathway gliomas (OPG). The NF1 tumor suppressor gene product, neurofibromin, functions as a Ras GTPase-activating protein, and has been proposed to regulate cell growth by inhibiting Ras activity. Recent studies from our laboratory have shown that neurofibromin also regulates the mammalian target of rapamycin activity in a Ras-dependent fashion, and that the rapamycin-mediated mammalian target of rapamycin inhibition ameliorates the Nf1-/- astrocyte growth advantage. Moreover, Nf1-deficient astrocytes exhibit increased protein translation. As part of a larger effort to identify protein markers for NF1-associated astrocytomas that could be exploited for therapeutic drug design, we did an objective proteomic analysis of the cerebrospinal fluid from genetically engineered Nf1 mice with optic glioma. One of the proteins found to be increased in the cerebrospinal fluid of OPG-bearing mice was the eukaryotic initiation factor-2alpha binding protein, methionine aminopeptidase 2 (MetAP2). In this study, we show that Nf1 mouse OPGs and NF1-associated human astrocytic tumors, but not sporadic pilocytic or other low-grade astrocytomas, specifically expressed high levels of MetAP2. In addition, we show that Nf1-deficient astrocytes overexpress MetAP2 in vitro and in vivo, and that treatment with the MetAP2 inhibitor fumagillin significantly reduces Nf1-/- astrocyte proliferation in vitro. These observations suggest that MetAP2 is regulated by neurofibromin, and that MetAP2 inhibitors could be potentially employed to treat NF1-associated tumor proliferation.

Characterization of an aminopeptidase in cerebrospinal fluid. Structure elucidation of enzyme hydrolysis products of synthetic methionine-enkephalin by reversed-phase high-performance liquid chromatography and mass spectrometry.

An aminopeptidase was found in canine cerebrospinal fluid via the presence of two products: Y, which has an [M + H]+ ion at m/z 182; and GGFM, which has an [M + H]+ ion at m/z 411. The linked scan at a constant ratio of the magnetic field to the electric field of the GGFM [M + H]+ ion at m/z 411 generates product ions at m/z 120, 150, 266, 297, 354, 357, and 411. That aminopeptidase was bestatin-sensitive (BSAP = bestatin-sensitive aminopeptidase), and had a half-time for disappearance of 60 min, maximum velocity of 1.08 nmol ml-1 min-1, and Michaelis constant of 0.26 nM.

N-terminal degradation of low molecular weight opioid peptides in human cerebrospinal fluid.

Opioid peptides are present in human cerebrospinal fluid (CSF), and their levels are reported to change in some pathologic conditions. However, less is known about their degradation in CSF. In the present study, human CSF was found to contain aminopeptidase activity which hydrolyzed alanyl-, leucyl- and arginyl-naphthylamides in a ratio of 100:28:27. Twelve CSF samples hydrolyzed alanyl-2-naphthylamide and degraded Met5-enkephalin (N-terminal hydrolysis) at rates of 188 +/- 38 and 420 +/- 79 pmol/min/mL respectively. Further, the distribution of alanyl-naphthylamidase activity in individual samples (39-437 pmol/min/mL) was closely correlated with that of Met5-enkephalin degradation (37-833 pmol/min/mL). Both alanyl-naphthylamidase and enkephalin degradation were optimal at pH 7.0 to 7.5 and were inhibited by aminopeptidase inhibitors amastatin (IC50 = 20 nM), bestatin (4-7 microM) and puromycin (30-35 microM). Conversely, degradation was unaffected by inhibitors of neutral endopeptidase (phosphoramidon), carboxypeptidase N (MERGETPA) or angiotensin converting enzyme (captopril). The Km of Met5-enkephalin for the CSF aminopeptidase activity was 201 +/- 19 microM (N = 4). Rates of hydrolysis of the Tyr1-Gly2 bond of larger opioid peptides decreased with increasing peptide length. Pooled, concentrated CSF hydrolyzed Leu5-enkephalin, dynorphin A fragments [1-7], [1-10] and [1-13] and dynorphin A at rates of 2.05 +/- 0.27, 1.27 +/- 0.18, 0.94 +/- 0.06, 0.55 +/- 0.14 and 0.16 +/- 0.03 nmol/min/mL respectively. When analyzed by rocket-immunoelectrophoresis against antisera to aminopeptidase M (EC 3.4.11.2), the concentrated CSF formed an immunoprecipitate which could be stained histochemically for alanyl-naphthylamidase activity. These data are consistent with a significant role for aminopeptidase M activity in the degradation of low molecular weight opioid peptides in human CSF.

Aminopeptidase in human CSF which degrades delta-sleep inducing peptide (DSIP).

This study describes the purification and characterization of an aminopeptidase from human cerebrospinal fluid capable of degrading delta-sleep-inducing-peptide (DSIP). The enzyme has an apparent molecular weight of approximately 80,000 dalton. It is sensitive towards amastatin, bestatin and EDTA and is optimally active at neutral pH. The recovered enzyme was also found to degrade other neuropeptides, e.g., the enkephalins.

[A study of the leucine enkephalin like substance and enkephalin degrading enzyme activity in human cerebrospinal fluid].

Leucine enkephalin like substance (Leu-Enk) and enkephalin degrading enzyme activity (EDEA) in cerebrospinal fluid (CSF) of three patients suffering from pain were measured. As compared with control values before treatments, the values of Leu-Enk in CSF decreased, and those of EDEA increased in all the patients. After the treatment, two patients were fortunately cured of pain, and their two values returned to normal. But the other patient could not recover from severe pain, and their values remained abnormal. It is concluded that Leu-Enk and enkephalin degrading enzyme in CSF may play an important role in regulation of pain sensitivity.

[A new highly active proteolytic myelin enzyme and its possible role in the mechanism of the demyelinating process]. / Novyi vysokoaktivnyi proteoliticheskii ferment mielina i ego vozmozhnaia rol' v mekhanizme demieliniziruiushchego protsessa.

The study of the myelin in the rabbit brain and in biological fluids of patients with multiple sclerosis has revealed the presence of a new highly active proteolytic enzyme. The article describes the physicochemical properties of this enzyme. Its role in the pathogenesis of the demyelinizing process is emphasized: normally being coupled with an inhibitor the enzyme is released as a result of the demyelinizing process and becomes active. This leads to the destruction of myelin proteins. Possible factors of enzyme activation are considered. The authors discuss the significance of differences in the activities depending on the stage of the process and the course of multiple sclerosis.

Pyroglutamate aminopeptidase activity in human cerebrospinal fluid decreases with age.

The activity of pyroglutamate aminopeptidase, the major enzyme catalyzing thyrotropin-releasing hormone (TRH) metabolism in human CSF, decreased with age. This decrement is not due to age-dependent appearance of any enzyme inhibitor in CSF. The results of these studies underline the importance of using age-matched controls in assessing abnormalities of TRH metabolism in CSF during disease states.

Metabolism of thyrotropin-releasing hormone in human cerebrospinal fluid. Isolation and characterization of pyroglutamate aminopeptidase activity.

Pyroglutamate aminopeptidase, which catalyzes metabolism of thyrotropin-releasing hormone (TRH) to cyclo(His-Pro), is the major enzyme of TRH metabolism in human CSF. The partially purified CSF pyroglutamate aminopeptidase has a pH optimum between 6.0 and 7.4, and a Km of 15.9 +/- 3.1 microM. A number of potential competitive inhibitors of the enzymatic activity were examined, of which luteinizing hormone-releasing hormone and bombesin were the most effective. An examination of the structure of various peptides that inhibit pyroglutamate aminopeptidase activity indicated that the enzyme generally prefers a substrate having amino-terminal pyroglutamic acid (pGlu) and a COOH-terminal that is either blocked or distant from amino-terminal pGlu. Heavy metals, EDTA and reducing agents inactivated the enyzme, whereas benzamidine, phenylmethylsulfonylfluoride, trypsin inhibitor and alkylating agents had little or no effect on the enzymatic activity. Thiol-oxidizing agent 5,5'-dithiobis(2-nitrobenzoic acid), however, considerally inhibited the enzymatic activity. We hypothesize that CSF pyroglutamate aminopeptidase may play a role in the biologic actions of TRH.

Partial purification of two distinct enkephalin-degrading aminopeptidases from human cerebrospinal fluid.

In human cerebrospinal fluid, aminopeptidase, dipeptidyl aminopeptidase, dipeptidyl carboxypeptidase, and carboxypeptidase which were capable of hydrolyzing enkephalins were detected. Among these enzymes, two distinct aminopeptidase, designated C-AP1 and C-AP2, were partially purified. These enzymes were not purified thoroughly, but the characteristics of C-AP2 were similar to those of an aminopeptidase purified from monkey brain. But the inhibitory activity of amastatin on C-AP2 was stronger, and that of substance P was negligible. On the other hand, characteristics of C-Ap1 were extremely differ from those of C-AP2 or an aminopeptidase purified from monkey brain. C-AP1 had an optimum pH more in the acidic range (the highest at pH 6.0) and was not inhibited by any of the protease inhibitor tested including bestatin and amastatin.