Abnormal development of hypoxanthine-guanine phosphoribosyltransferase-deficient CNS neuroblastoma.

Lesch-Nyhan syndrome encompasses a host of neurological symptoms, caused by a deficiency of the purine salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGPRT). How the absence of this enzymes activity affects development of the nervous system is unknown. In this study, we examined the ability of N2aTG, a HGPRT-deficient neuroblastoma and its HGPRT-positive counterpart to proliferate and differentiate at various densities. In summary, N2aTG cells proliferated less and differentiated more than N2a cells, with the former cells exhibiting enhanced sensitivity to the effects of low-density culture. Given the homogeneity of this neuroblastoma cell line and its use in studies of neuronal development, the present study indicates that N2aTG cells may prove a suitable in vitro model for the study of non-dopaminergic neuronal development in Lesch-Nyhan syndrome.     

Lesch–Nyhan disease and the basal ganglia

The purpose of this review is to summarize emerging evidence that the neurobehavioral features of Lesch–Nyhan disease (LND), a developmental disorder caused by congenital deficiency of the purine salvage enzyme hypoxanthine–guanine phosphoribosyltransferase (HPRT), may be attributable to dysfunction of the basal ganglia. Affected individuals have severe motor disability described by prominent extrapyramidal features that are characteristic of dysfunction of the motor circuits of the basal ganglia. They also display disturbances of ocular motility, cognition, and behavioral control that may reflect disruption of other circuits of the basal ganglia. Though neuropathologic studies of autopsy specimens have revealed no obvious neuroanatomical abnormalities in LND, neurochemical studies have demonstrated 60–90% reductions in the dopamine content of the basal ganglia. In addition, recent PET studies have documented significant reductions in dopamine transporters and [¹⁸F]fluorodopa uptake in the basal ganglia. These findings support the proposal that many of the neurobehavioral features of LND might be related to dysfunction of the basal ganglia.     

Cross-sensitization between footshock stress and apomorphine on self-injurious behavior and neostriatal catecholamines in a rat model of Lesch–Nyhan syndrome

The effects of footshock sensitization (priming), apomorphine (APO) priming and their combination on behavior and neostriatal and cortical catecholamines were examined in adult rats which had neonatally received bilateral intracerebroventricular injections with 6-hydroxydopamine (6-OHDA; a model of Lesch–Nyhan syndrome (LNS)) or vehicle (unlesioned rats). Lesioned (6-OHDA-treated) rats displayed self-biting (SB; 7/20 rats) and self-injurious behavior (SIB; 1/20 rats) during APO priming, but not during footshock priming. During subsequent acute cumulative APO dosing, 20–30% of lesioned rats primed with APO alone or footshock alone displayed SB and SIB. However, SB and SIB incidence in APO+footshock-primed lesioned rats was nearly tripled. Dopamine (DA) synthesis, metabolism and extracellular concentrations (disposition) in unlesioned rats and in cortices of lesioned animals were unaffected by priming. In lesioned rats primed with APO alone or footshock alone, only neostriatal 3-methoxytyramine (3-MT) was significantly increased. However, neostriatal DA and metabolite concentrations (and norepinephrine (NE)) were all significantly elevated in lesioned rats primed with both APO and footshock. These results confirm that neonatal 6-OHDA-induced neostriatal catecholamine depletion can be antagonized by experiential change, suggest that behavioral and neurochemical cross-sensitization between APO and footshock in such rats is unidirectional and support the view that stress can exacerbate the incidence of SIB in LNS.     

Hypoxanthine impairs morphogenesis and enhances proliferation of a neuroblastoma model of Lesch Nyhan syndrome

Extracellular purines have essential roles in neuronal development; hence, disruptions in their metabolism as reported in Lesch Nyhan syndrome (LNS) could result in developmental abnormalities. The deficiency of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) in LNS leads to increased hypoxanthine and uric acid production. We have reported that HGPRT-deficient B103-4C neuroblastoma, a neuronal model of LNS, proliferated less and differentiated more than their HGPRT-positive B103 counterparts. Here, we sought to determine whether differences in proliferation and differentiation would occur when these cells were cultured in the presence of hypoxanthine or in a hypoxanthine-/serum-free chemically defined media (NBMN2). In media with 1% serum, hypoxanthine (50 microM) significantly increased the proliferation of both cell lines with a greater effect on B103-4C cells. In 1% serum media, hypoxanthine increased differentiation of B103 but decreased B103-4C differentiation. In NBMN2, B103 proliferated far more than B103-4C, but both cell types differentiated to the same extent. These results are interpreted to suggest that elevated levels of central nervous system (CNS) hypoxanthine as reported in LNS may affect neuronal development, and to implicate hypoxanthine and abnormal neuronal development as causative factors in the etiology of LNS.     

Estrogen–BDNF interactions: Implications for neurodegenerative diseases

Since its’ discovery over 20 years ago, BDNF has been shown to play a key role in neuronal survival, in promoting neuronal regeneration following injury, regulating transmitter systems and attenuating neural-immune responses. Estrogen’s actions in the young and mature brain, and its role in neurodegenerative diseases in many cases overlaps with those observed for BDNF. Reduced estrogen and BDNF are observed in patients with Parkinson’s disease and Alzheimer’s disease, while high estrogen levels are a risk factor for development of multiple sclerosis. Estrogen receptors, which transduce the actions of estrogen, colocalize to cells that express BDNF and its receptor trkB, and estrogen further regulates the expression of this neurotrophin system. This review describes the distribution of BDNF and trkB expressing cells in the forebrain, and the roles of estrogen and the BDNF–trkB neurotrophin system in Parkinson’s disease, Alzheimer’s disease and multiple sclerosis.     

Interaction between optineurin and Rab1a regulates autophagosome formation in neuroblastoma cells

Optineurin (OPTN) is an autophagy receptor protein that has been implicated in glaucoma and amyotrophic lateral sclerosis. OPTN‐mediated autophagy is a complex process involving many autophagy‐regulating proteins. Autophagy plays a critical role in removing damaged organelles, intracellular pathogens, and protein aggregates to maintain cellular homeostasis. We identified Ypt1 as a novel interaction partner of OPTN by performing a large‐scale yeast‐human two‐hybrid assay. Coimmunoprecipitation assay showed that OPTN interacted with Rab1, the mammalian homolog of yeast Ypt1, in N2a mouse neuroblastoma cell line. We confirmed this interaction by confocal microscopy showing intracellular colocalization of the two proteins. We observed that a zinc finger domain of OPTN is important for Rab1a binding. Rab1a activity is also required for the binding with OPTN. The role of the OPTN‐Rab1a complex in neuronal autophagy was determined by measuring the translocation of microtubule‐associated protein light chain 3–EGFP to autophagosomes. In N2a cells, OPTN‐induced autophagosome formation was inhibited by Rab1a knockdown, indicating the important role of OPTN‐Rab1a interaction in neuronal autophagy processes. Similarly, in N2a cells overexpressing Rab1a, serum starvation–induced formation of autophagosome was enhanced, while OPTN knockdown reduced the Rab1a‐induced autophagy. These results show that the OPTN‐Rab1a complex modulates autophagosome formation in neuroblastoma cells.     

Vesicular trafficking and secretion of matrix metalloproteinases-2, -9 and tissue inhibitor of metalloproteinases-1 in neuronal cells

Matrix metalloproteinases (MMPs) are endopeptidases that cleave matrix, soluble and membrane-bound proteins and are regulated by their endogenous inhibitors the tissue inhibitors of MMPs (TIMPs). Nothing is known about MMP/TIMP trafficking and secretion in neuronal cells. We focussed our attention on the gelatinases MMP-2 and MMP-9, and their inhibitor TIMP-1. MMPs and TIMP-1 fused to GFP were expressed in N2a neuroblastoma and primary neuronal cells to study trafficking and secretion using real time video-microscopy, imaging, electron microscopy and biochemical approaches. We show that MMPs and TIMP-1 are secreted in 160–200 nm vesicles in a Golgi-dependent pathway. These vesicles distribute along microtubules and microfilaments, co-localise differentially with the molecular motors kinesin and myosin Va and undergo both anterograde and retrograde trafficking. MMP-9 retrograde transport involves the dynein/dynactin molecular motor. In hippocampal neurons, MMP-2 and MMP-9 vesicles are preferentially distributed in the somato-dendritic compartment and are found in dendritic spines. Non-transfected hippocampal neurons also demonstrate vesicular secretion of MMP-2 in both its pro- and active forms and gelatinolytic activity localised within dendritic spines. Our results show differential trafficking of MMP and TIMP-1-containing vesicles in neuronal cells and suggest that these vesicles could play a role in neuronal and synaptic plasticity.     

Abnormal adenosine and dopamine receptor expression in lymphocytes of Lesch–Nyhan patients

Self-injurious behavior is the most outstanding feature of Lesch–Nyhan syndrome and has recently been ascribed to an obsessive–compulsive behavior. Lesch–Nyhan syndrome results from the complete enzyme deficiency of hypoxanthine–guanine phosphoribosyl transferase (HPRT) but the link between abnormal purine metabolism and its neurological and behavioral manifestations remains largely unknown. Previous studies led us to hypothesize that adenosine and dopamine receptor expression could be altered in HPRT-deficient cells. To test this hypothesis, we examined mRNA expressions of adenosine (ADORA2A and ADORA2B) and dopamine receptors (DRD1 and DRD2 like), and dopamine transporter (DAT1) in peripheral blood lymphocytes (PBLs) from Lesch–Nyhan patients. We also examined the influence of hypoxanthine in these expressions. As compared to normal PBLs, both ADORA2A and DRD5 expression were abnormal in PBLs from Lesch–Nyhan patients. In contrast, DAT1 expression was similar to control values in HPRT deficient PBLs. These results indicate an abnormal adenosine and dopamine receptor expression in HPRT-deficient cells and suggest disrupted adenosine and dopamine neurotransmission may have a significant role in the pathogenesis of the neurological manifestations of Lesch–Nyhan syndrome.     

Fixed-ratio discrimination training as replacement therapy in Parkinson''s disease: studies in a 6-hydroxydopamine-treated rat model

Severe 6-hydroxydopamine (6-OHDA)-induced neostriatal dopamine (DA) depletion is generally held to be irreversible. Adult rats administered 6-OHDA soon after weaning, or neonatally, respectively model Parkinson's disease (PD) and Lesch–Nyhan syndrome (LNS). Prior studies in our laboratory indicate that prolonged training on incrementally more difficult fixed-ratio (FR) discriminations can reverse ‘irreversible' 6-OHDA-induced neostriatal DA depletion in adult LNS rats. The present study evaluated the effects of such training on neostriatal DA depletion and its functional consequences in adult PD and control (vehicle-injected) rats. After recovery from 6-OHDA-induced hypophagia, rats were sacrificed to assess neostriatal DA depletion magnitude, or were food-deprived and either subjected to food-maintained operant FR discrimination training or allowed to remain in their home cages. 6-OHDA treatment antagonized amphetamine (AMP)-induced increases in brief rearing behavior and locomotor activity in 3-month-old PD rats prior to training, and reduced operant response rates throughout training without affecting learning rates. One week after training, AMP-increased locomotor and brief-rearing frequencies were augmented in all groups except trained controls, and the prior inhibitory effect of 6-OHDA treatment on AMP-increased behavioral frequencies was essentially eliminated. Cumulative apomorphine (APO) dose–effect curve (0.1–3.2 mg/kg) construction 3 weeks post-training revealed that 6-OHDA treatment abolished APO-induced intense licking behavior. However, training eliminated the hyperresponsiveness of 6-OHDA-treated rats to the locomotor- and brief-rearing stimulant effects of APO but did not affect the depletion of neostriatal DA. Nevertheless, 6-OHDA-induced increases in neostriatal DOPAC/DA and HVA/DA ratios were normalized by age/food-deprivation while that of 3MT/DA was not. These findings suggest that training reduces the functional responsiveness of at least some central DA receptors, FR discrimination training could be a useful adjunct to PD replacement therapy and that the neostriatal DA-repleting action of training in 6-OHDA-treated rats depend on the age at which 6-OHDA is administered.     

Release of brain–type and heart–type fatty acid–binding proteins in serum after acute ischaemic stroke

This study aimed at an analysis of the release of Braintype and Heart–type Fatty Acid– Binding Proteins (B–FABP and HFABP) in acute ischaemic stroke and their potential value as neurobiochemical markers of brain damage.We investigated 42 consecutive patients admitted within 6 hours after ischaemic stroke. Serial venous blood samples were taken hourly between 1 to 6 hours, and at 12, 18, 24, 48, 72, 96, and 120 hours after stroke onset. In all patients lesion topography was assessed and infarct volume was calculated. The neurological deficit was quantified by the National Institutes of Health stroke scale score, and functional outcome was assessed with the modified Rankin Scale 3 months after stroke.H–FABP and B–FABP concentrations showed peak values already 2 to 3 hours after stroke onset and remained elevated up to last measurements at 120 hours.Unlike BFABP, early H–FABP concentrations were significantly associated with the severity of the neurological deficit and the functional outcome. High H–FABP release was associated with large infarction on CT.Our study shows for the first time quantitative data of serum BFABP and H–FABP being elevated early in acute ischaemic stroke indicating that especially H–FABP might have the potential to be a rapid marker of brain damage and clinical severity. As both FABPs indicate damage to neuronal and glial tissue but are not specific for cerebral infarction, further investigations are needed to better understand the prolonged release of both in ischaemic stroke which is in contrast to the transient increase after myocardial infarction and can not be explained by their renal extraction.     

Long-term cognitive outcome of hemiconvulsion-hemiplegia-epilepsy syndrome affecting the left cerebral hemisphere

Long-term cognitive outcome following hemiconvulsion–hemiplegia–epilepsy (HHE) syndrome has been poorly studied, with little attention to the implications of side of involvement in HHE. This retrospective study describes language lateralization and cognitive performance in five patients with HHE syndrome affecting the left cerebral hemisphere. All of the patients had to have intracarotid sodium amytal testing (IAT) to be included in this study. The mean age of the patients was 30.2 years (range: 13–50). All patients had their hemiconvulsive seizures before age 1½ years (range: 6–13 months). All patients had right-sided hemiatrophy of the body, left mesial temporal sclerosis, and seizures originating from the left temporal lobe. The habitual seizures began at a mean age of 4.5 years (range: 1.5–12 years). Performance on tests of intelligence, verbal memory, and visual memory was examined. Language was represented in the right cerebral hemisphere in three patients, the left hemisphere in one patient, and both hemispheres with predominance on the right side in the fifth patient. Intellectual functioning was in the borderline to extremely low range among the patients with right hemispheric or bilateral representation for language. These patients were variably impaired on measures of verbal and visual memory. The patient with left hemispheric representation for language performed in the average range on tests of intellectual functioning and verbal memory, whereas scores on visual memory were variable. This study demonstrated that reorganization of language to the right cerebral hemisphere or its bilateral representation is common in patients with HHE syndrome affecting the left cerebral hemisphere, and is associated with poor cognitive outcome.     

Role of mitofusin 2 mutations in the physiopathology of Charcot–Marie–Tooth disease type 2A

Charcot–Marie–Tooth disease (CMT) is the most common form of hereditary peripheral neuropathy. The main axonal form of CMT, CMT2A, preferentially affects peripheral neurons with the longest neurites. CMT2A has been recently linked to mutations in the mitofusin 2 (Mfn2) gene. Mfn2 participates in mitochondrial fusion a process that together with mitochondrial fission, contributes to mitochondrial morphology. Many hypotheses have been postulated to understand how mutations in Mfn2 lead to CMT2A. In this review, we will describe the physiological role of Mfn2, the pathophysiology of CMT2A and current hypotheses about the deleterious role of mutant Mfn2 in neuronal function.     

PEN–2 gene mutation in a familial Alzheimer’s disease case

Genetic evidence indicates a central role of cerebral accumulation of β–amyloid (Aβ) in the pathogenesis of Alzheimer’s disease (AD). Beside presenilin 1 and 2, three other recently discovered proteins (Aph 1, PEN 2 and nicastrin) are associated with γ–secretase activity, the enzymatic complex generating Aβ. Alterations in genes encoding these proteins were candidates for a role in AD. The PEN 2 gene was examined for unknown mutations and polymorphisms in sporadic and familial Alzheimer patients. Samples from age–matched controls (n = 253), sporadic AD (SAD, n = 256) and familial AD (FAD, n = 140) were screened with DHPLC methodology followed by sequencing. Scanning the gene identified for the first time a missense mutation (D90N) in a patient with FAD. Three intronic polymorphisms were also identified, one of which had a higher presence of the mutated allele in AD subjects carrying the allele ε4 of apolipoprotein E than controls. The pathogenic role of the PEN–2 D90N mutation in AD is not clear, but the findings might lead to new studies on its functional and genetic role.*These two authors contributed equally to the paper.     

Clinical effects of COX–2 inhibitors on cognition in schizophrenia

An activation of pro–inflammatory cytokines in the central nervous system is associated with cognitive disturbances. This process is mediated by prostaglandins and cyclo–oxygenase–2 (COX–2). COX–2 inhibitors have been suggested to show beneficial effects in disorders associated with cognitive disturbance, although clinical effects on cognition have not been shown until today. Data from a schizophrenia study were reevaluated under the aspect whether an effect on the positive and negative syndrome scale (PANSS) factor cognition can be observed during therapy with the COX–2 inhibitor celecoxib add on to risperidone in comparison to riperidone alone. Beside the effect on the PANSS total score, the effect on the cognition factor was the most pronounced using the analysis of covariance compared to all other factors of the PANSS (p <0.06). Although suggestions of basic research led to an expected therapeutic effect of COX–2 inhibitors on cognition, this effect could not yet be shown clinically. In schizophrenia, the effect on cognition contributes to the therapeutic effect of COX–2 inhibitors.     

The neonate-6-hydroxydopamine-lesioned rat: a model for clinical neuroscience and neurobiological principles

In 1973, a technique of administering 6-hydroxydopamine (2,4,5-trihydroxyphenylethylamine) intracisternally to neonate rats was introduced to selectively reduce brain dopamine (neonate-lesioned rat). This neonate treatment proved unique when compared to rats lesioned as adults with 6-hydroxydopamine—prompting the discovery of differing functional characteristics resulting from the age at which brain dopamine is reduced. A realization was that neonate-lesioned rats modeled the loss of central dopamine and the increased susceptibility for self-injury in Lesch–Nyhan disease, which allowed identification of drugs useful in treating self-injury in mentally retarded patients. The neonate-lesioned rat has also been proposed to model the hyperactivity observed in attention-deficit hyperactivity disorder. Because the neonate-lesioned rat exhibits enhanced sensitization to repeated NMDA receptor antagonist administration and has functional changes characteristic of schizophrenia, the neonate lesioning is believed to emulate the hypothesized NMDA hypofunction in this psychiatric disorder. Besides modeling features of neurological and psychiatric disorders, important neurobiological concepts emerged from pharmacological studies in the neonate-lesioned rats. One was the discovery of coupling of D₁/D₂-dopamine receptor function. Another was the progressive increase in responsiveness to repeated D₁-dopamine agonist administration referred to as “priming” of D₁-dopamine receptor function. Additionally, a unique profile of signaling protein expression related to neonate reduction of dopamine has been identified. Thus, from modeling characteristics of disease to defining adaptive mechanisms related to neonatal loss of dopamine, the neonate-lesioned rat has had a persisting influence on neuroscience. Despite an extraordinary legacy from studies of the neurobiology of this treatment, a host of unknowns remain that will inspire future investigations.     

Cyclooxygenase 2 mRNA expression in rat brain after peripheral injection of lipopolysaccharide

Inducible cyclooxygenase 2 (COX 2) converts arachidonic acid to prostaglandins, which are thought to mediate various peripheral lipopolysaccharide (LPS)-induced central effects, including generation of fever and activation of the hypothalamic–pituitary–adrenal axis. To localize prostaglandin production in the brain following peripheral LPS administration, COX 2 mRNA expression was examined by in situ hybridization histochemistry in rats injected intraperitoneally (i.p.) or intravenously (i.v.) with various doses of LPS or saline. Constitutive expression of COX 2 mRNA was found in neurons of cortex, hippocampus, and amygdala, but not in cells of the blood vessels. COX 2 mRNA levels were not altered in saline-injected animals as compared to non-injected controls. In LPS-injected animals, no consistent changes of neuronal COX 2 mRNA expression were observed. COX 2 mRNA expression appeared ex novo at 0.5-h post-injection in cells closely associated with blood vessels, however, ex novo labeling of the number of labeled cells increased to a peak at 2 h and subsided gradually to basal levels by 24 h. Initially, labeling was observed in cells comprising major surface-lying blood vessels and meninges. Later, vascular and perivascular cells associated with smaller penetrating blood vessels were labeled. This pattern of COX 2 mRNA induction is independent of the route and dose of the LPS injection. The induced COX 2 mRNA producing cells are identified as endothelial and leptomeningeal cells. Changes in COX 2 mRNA expression were not observed in circumventricular organs. These results suggest that peripheral LPS induces a rapid increase in COX 2 production throughout the vasculatures of the brain, which could affect the neuronal activity of widespread brain regions by elevating the levels of prostaglandins.     

MEK/ERKs signaling is essential for lithium-induced neurite outgrowth in N2a cells

Lithium, a drug used for the treatment of bipolar disorder, has been shown to affect different aspects of neuronal development such as neuritogenesis, neurogenesis and survival. The underlying mechanism responsible for lithium's influence on neuronal development, however, still remains to be elucidated. In the present study, we demonstrate that lithium increases the phosphorylation of extracellular-signal regulated kinases (ERKs) and protein kinase B (Akt) and promotes neurite outgrowth in mouse N2a neuroblastoma cells (N2a). The inactivation of mitogen-activated protein kinase kinase (MEK)/ERKs signaling with a MEK inhibitor inhibits neurite outgrowth, but it enhances Akt activation in lithium-treated N2a cells. Furthermore, the inactivation of phosphoinositide-3-kinase (PI3K)/Akt signaling with a PI3K inhibitor increases both lithium-induced ERKs activation and lithium-induced neurite outgrowth. Taken together, our study suggests that lithium-induced neurite outgrowth in N2a cells is regulated by cross-talk between the MEK/ERKs and PI3K/Akt pathways and requires the activation of the MEK/ERKs signaling.     

17p duplicated Charcot–Marie–Tooth 1A

The most frequent type of Charcot–Marie–Tooth (CMT) neuropathy is that associated with the 17p11.2–p12 chromosome duplication, whose characteristics have been well described in European and North American populations. In this study, we analyzed a Brazilian population exhibiting the mutation, found in 57 patients from 42 families (79%) of a cohort of 53 families with demyelinating CMT. Almost 20% of the duplicated cases were sporadic. In 77% of the duplicated families the mutation event occurred in the hot spot area of the CMT1A–Rep region. Forty–five percent of patients were females, 84% were Caucasians and 13% of African descent. Distal limb weakness was the most frequent abnormality, appearing in 84% of patients, although uncommon manifestations such as severe proximal weakness, floppy baby syndrome, diaphragmatic weakness and severe scoliosis were also observed. One patient was wheelchair–bound, and three suffered severe hand weakness. Sensory abnormalities were detected in 84% of the cases, but 80% were unaware of this impairment. Twelve patients complained of positive sensory manifestations such as pain and paresthesias. Progression was reported by 40%. Motor conduction velocities in the upper limbs were always less than 35 m/s, and less than 30.4 m/s in the peroneal nerve. The findings of this study expand the clinical spectrum of the disease.     

Reflections on the brainstem dysfunction in neurologically disabled children

This article deals with the neurological basis of brainstem-related symptoms in disabled children. Synaptic interactions of respiratory and swallowing centers, which are briefly reviewed in this study, highlight the significance of the nucleus of solitary tract (NTS) in the stereotyped motor events. Coordination mechanisms between these two central pattern generators are also studied with a focus on the inhibitory action of decrementing expiratory neurons that terminate the inspiratory activity and become activated during swallowing. Dorsal brainstem lesions in hypoxic–ischemic encephalopathy (HIE) affect the area including NTS, and result in symptoms of apneusis, facial nerve paresis, dysphagia, gastroesophageal reflux, and laryngeal stridor. Leigh syndrome patients with similar distributions of medullary lesions show increased sighs, post-sigh apnea, hiccups, and vomiting in addition to the symptoms of HIE, suggesting pathologically augmented vagal reflex pathways. The present article also discusses the pathophysiology of laryngeal dystonia in xeroderma pigmentosum group A, self-mutilation in Lesch–Nyhan syndrome, and sudden unexpected death in Fukuyama congenital muscular dystrophy. Close observation and logical assessment of brainstem dysfunction symptoms should be encouraged in order to achieve better understanding and management of these symptoms in disabled children.     

Application of quantitative LC–MS surrogate peptide methodology in the analysis of the amyloid beta peptide (Aβ) biosynthetic intermediate protein APP–βCTF

An area of current research in Alzheimer's disease (AD) is the biosynthetic pathway of amyloid beta peptides (Aβ) via consecutive proteolytic cleavages of the amyloid beta precursor protein (APP) by BACE and γ-secretase enzymes. APP is first cleaved by BACE to form a C-terminal fragment APP–βCTF, or also called C99, which then undergoes further cleavage by γ-secretase to form Aβ. Inhibitors of γ-secretase have been observed to yield a so-called ‘Aβ rise’ phenomenon whereby low inhibitor concentrations result in an increase in Aβ levels while high inhibitor concentrations result in lower Aβ levels. A previous report from our labs indicated that this phenomenon was related to ratios of APP–βCTF substrate relative to γ-secretase enzyme. A quantitative Western blot analysis was used with a recombinant C100 protein as calibration standards to assess the relationship of APP–βCTF, γ-secretase enzyme and various inhibitors resulting in the ‘Aβ rise’. An on-line liquid chromatography mass spectrometry (LC–MS) method employing the ‘surrogate peptide’ methodology was developed to accurately quantify the recombinant C100 used in the Western blot analyses. The surrogate peptide approach utilizes tryptic digestion of the protein to stoichiometrically yield a unique peptide fragment, in this case ^C100Aβ17–28 (LVFFAEDVGSNK) that can be readily detected by LC–MS. The absolute quantitative assessment of C100 was accomplished using synthetic Aβ17–28 to generate calibration curves over a 0.001–1 μM range and 15N isotopically labeled Aβ1–40 as the internal standard for enzymatic digestion and its proteolytic peptide [15N]-Aβ17–28 for the analysis.