Antimo D’Aniello

Amino acids and transaminases activity in ventricular CSF and in brain of normal and Alzheimer patients.

The present study was conducted to determine the concentration of amino acids in the cerebrospinal spinal fluid (CSF) and the activities of two tramsaminases: glutamic oxaloacetate transaminase (GOT) and glutamic pyruvate transaminase (GPT) in human Alzheimer disease (AD) and normal brain. L-glutamic acid, L-glutamine and L-alanine are the most abundant amino acids in the CSF (50-55% of total amino acids). L-glutamine occurs at much higher levels in Alzheimer CSF compared to the normal CSF (229+/-91.8 nmol/ml in AD versus 107+/-47.2 nmol/ml in normal; P=0.0041). In contrast, L-aspartate occurs at significantly lower concentrations in Alzheimer CSF than normal CSF (46.1+/-25.7 nmol/ml in Alzheimer versus 95.2+/-52.6 nmol/ml in normal; P=0.020). In Alzheimer brain (frontal, parietal and occipital cortices) GOT is present at significantly higher activities than in normal brain cortices (about 1.5 times higher; P<0.01). No significant differences for GPT activity occurred between normal and AD brain. Since CSF receives amino acids from brain tissues, and since GOT catalyzes the conversion of L-aspartate to L-glutamate, the higher concentrations of L-glutamine (which is derived from L-glutamate), and the lower concentrations of L-aspartate found in Alzheimer CSF could be considered as a consequence of the higher activity of GOT that occurs in Alzheimer brain.

Plasma Protein Aspartyl Damage Is Increased in Hemodialysis Patients: Studies on Causes and Consequences

Plasma proteins in hemodialysis patients display a significant increase in deamidated/isomerized Asx (asparagine and aspartic acid) content, a marker of protein fatigue damage. This has been linked to the toxic effects of hyperhomocysteinemia in uremic erythrocytes; however, treatment aimed at abating homocysteine levels did not lead to significant reductions in plasma protein damage. The hypothesis that lack of reduction in protein damage could be due to protein increased intrinsic instability, as result of interference with the uremic milieu rather than to hyperhomocysteinemia, was put forward. The deamidated/isomerized Asx content of normal plasma incubated with several uremic toxins for 24 h, 72 h, and 7 d was measured, identifying a group of toxins that were able to elicit this kind of damage. Uremic toxins were also incubated with purified human albumin, and dose-response experiments with the two most toxic agents in terms of protein damage (guanidine and guanidinopropionic acid) were carried out. The effect of the hemodialysis procedure on protein damage was evaluated. For investigating also the consequences of these alterations, human albumin was treated in vitro to produce an increase in deamidated/isomerized Asx residues, and the effects of albumin deamidation on protein binding were evaluated. Among the uremic toxins that are able to elicit protein damage, guanidine produced a dose-dependent increase in protein damage. No difference was found after a hemodialysis session. Deamidated albumin shows normal binding capacity to warfarin, salicylic acid, or diazepam but reduced binding to homocysteine. In conclusion, uremic toxins, especially guanidine, display an ability to induce significant protein damage, which can in turn have functional consequences.

Stimulation of androgen production by d-aspartate through the enhancement of StAR, P450scc and 3β-HSD mRNA levels in vivo rat testis and in culture of immature rat Leydig cells

Previous studies have shown a role of d-aspartic acid (d-Asp) in testicular steroidogenesis. Here, we evaluated the effects of d-Asp on androgen production and on expression levels of mRNAs encoding specific steroidogenic key molecules. d-Asp was endogenously present in adult rat testis and its content paralleled to serum luteinizing hormone (LH) and, local and circulating androstenedione and testosterone levels. In vivod-Asp administration induced serum LH release, causing an indirect increase of androstenedione and testosterone levels by enhancing steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase/D5-D4 isomerases (3β-HSD) mRNA levels. The direct endocrine role of d-Asp was evaluated using cultured immature Leydig cells (ILCs) obtained from 35days old rats. Cytoplasm and nucleus of ILCs localized d-Asp, while StAR marked the cytoplasm only. After 12h from d-Asp in vitro administration, ILCs resulted intensely d-Asp stained, and StaR protein level, evaluated by Western blotting, significantly increased. After 24h, significant androstenedione and testosterone syntheses were induced. At molecular level, d-Asp administration significantly increased StAR, P450scc and 3β-HSD mRNAs at 2, 4 and 12h, respectively. The temporal shift on relative mRNA expression levels indicated that d-Asp exerted its physiological role through sequential gene cascade activation of those molecules implicated in the synthesis of androgens. Conclusively, our findings demonstrated that d-Asp is a local messenger in testis and give a contribution in understanding the complexity of local endocrine regulation as well as the molecular events leading the acquisition to a steroidogenic competence by ILCs.

D-Aspartate affects secretory activity in rat Harderian gland: molecular mechanism and functional significance.

In this paper, the role of d-aspartate in the rat Harderian gland (HG) was investigated by histochemical, ultrastructural, and biochemical analyses. In this gland, substantial amounts of endogenous d-Asp were detected, along with aspartate racemases that convert d-Asp to l-Asp and vice versa. We found that the gland was capable of uptaking and accumulating exogenously administered d-Asp. d-Asp acute treatment markedly increased lipid and porphyrin secretion and induced a powerful hyperaemia in inter-acinar interstitial tissue. Since d-Asp is known to be recognized by NMDA receptors, the expression of such receptors in rat HG led us to the hypothesis that d-Asp acute treatment induced the activation of the extracellular signal-regulated protein kinase (ERK) and nitric oxide synthase (NOS) pathways mediated by NMDA. Interestingly, as a result of enhanced oxidative stress due to increased porphyrin secretion, the revealed activation of the stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) pro-apoptotic pathway was probably triggered by the gland itself to preserve its cellular integrity.

Elevated forebrain excitatory l-glutamate, l-aspartate and d-aspartate in the Naples high-excitability rats

The Naples high-excitability (NHE) rats are thought to model the mesocortical variant of attention-deficit hyperactivity disorder (ADHD). The aim of this study was to investigate forebrain level of L-glutamate, L-aspartate and D-aspartate, in NHE vs. Naples random bred (NRB) control rats. Thus, prepuberal NHE and NRB rats were daily handled in the 5th and 6th week of postnatal life. Then rats were exposed to two spatial novelties i.e. a Làt and a Olton maze for 10 min. Amino acids were detected by HPLC in the prefrontal cortex (PFC), striatum (STR), hippocampus (HPC) and hypothalamus (HYP). Results indicate that all amino acids were higher in NHE than in NRB rats. This, in turn, may explain the behavioural hyperactivity and attention deficit of this animal model of ADHD.

d-Aspartic acid ameliorates painful and neuropsychiatric changes and reduces β-amyloid Aβ1-42 peptide in a long lasting model of neuropathic pain

Depressive symptoms and other neuropsychiatric dysfunctions are common in neurodegenerative disorders, including chronic pain and dementia. A correlation between the β-amyloid protein accumulation and the development of depression has been suggested, however the underlying mechanisms are unknown. d-Aspartate (d-Asp) is a free d-amino acid found in the mammalian brain and involved in neurological and psychiatric processes, such as cognition and affective disorders. In this study we have investigated the effects of a repeated treatment with d-Asp in a long-lasting (12 months) model of neuropathic pain, the spared nerve injury (SNI), in mice. Specifically, we evaluated i) the pain sensitivity and related emotional/cognitive dysfunctions induced by SNI, ii) possible changes in the β-amyloid protein accumulation in specific brain regions involved in pain mechanisms ii) possible changes in steroids level in neuropathic animals with or without d-Asp in the same brain areas. SNI mice showed an increase of the insoluble form of Aβ1-42 at hippocampal level and displayed cognitive impairments, stereotypical and depressive-like behaviours. d-Asp treatment reduced abnormal behaviours and normalized the β-amyloid protein expression. Moreover, d-Asp dramatically increased steroids level measured in the prefrontal cortex and in the hippocampus. Our findings provide new insights into pain mechanisms and suggest a possible role of β-amyloid protein in neuropsychiatric dysfunctions associated with chronic pain.

Differential prepuberal handling modifies behaviour and excitatory amino acids in the forebrain of the Naples High-Excitability rats

Naples High-Excitability (NHE) rats model the mesocortical variant of Attention-Deficit Hyperactivity Disorder (ADHD). Recently, a high level of excitatory amino acids (EAA) has been found in the forebrain of NHE rats. The aim of this study was to verify the effect of postnatal stimulation in prepuberal rats on forebrain EAA. Thus, prepuberal NHE and Naples Random Bred (NRB) control rats were daily handled (PS) or they were left undisturbed throughout (NO-PS). One hour after the last stimulation, PS and NO-PS rats were exposed to a spatial novelty in a Làt-maze and one day later to a non-reinforced Olton maze. In both tests the horizontal (HA) and vertical (frequency - VA and duration of rearing - RD) components of behaviour indexed activity and non-selective attention (NSA). Moreover, in the Olton maze the position of the number of arms visited before first repetition (FE) and to criterion (NVTC), indexed selective spatial attention (SSA). Amino acids were detected by HPLC in prefrontal cortex (PFC), striatum (STR), hippocampus (HPC) and hypothalamus (HYP). Results indicate that (i) in the Làt-maze, only for HA, NO-PS NHE rats were more active than PS, (ii) in the Olton maze NO-PS rats of both lines showed shorter rearing durations than PS, (iii) EAA level was higher in NHE than in NRB rats and (iv) NO-PS vs. PS treatment increased level of EAA across the forebrain in both rat lines. In contrast in NHE NO-PS rats L-glutamate (L-Glu) decreased in HYP and L-aspartate (L-Asp) decreased in HPC. In conclusion, postnatal stimulation in prepuberal rats significantly affects forebrain excitatory amino acids and behaviour in NHE line. Thus EAA are modulated by genetic determinants and environmental (epigenetic) factors.

Sex hormone levels in the brain of d-aspartate-treated rats

d-Aspartate (d-Asp) is an endogenous amino acid present in the central nervous system and endocrine glands of various animal taxa. d-Asp is implicated in neurotransmission, physiology of learning, and memory processes. In gonads, it plays a crucial role in sex hormone synthesis. We have investigated the effects of chronic (30 days d-Asp drinking solution) and acute (i.p. injection of 2μmol/g bw d-Asp) treatments on sex steroid synthesis in rat brain. Furthermore, to verify the direct effect of d-Asp on neurosteroidogenic enzyme activities, brain homogenates were incubated with different substrates (cholesterol, progesterone, or testosterone) with or without the addition of d-Asp. Enzyme activities were measured by evaluating the in vitro conversion rate of (i) cholesterol to progesterone, testosterone, and 17β-estradiol, (ii) progesterone to testosterone and 17β-estradiol, (iii) testosterone to 17β-estradiol. We found that d-Asp oral administration produced an increase of approximately 40% in progesterone, 110% in testosterone, and 35% in 17β-estradiol. Similarly, the results of the acute experiment showed that at 30min after d-Asp treatment, the progesterone, testosterone, and 17β-estradiol levels increased by 29-35%, and at 8h they further increased by a 100% increment. In vitro experiments demonstrate that the addition of d-Asp to brain homogenate+substrate induces a significant increase in progesterone, testosterone and 17β-estradiol suggesting that the amino acid upregulates the local activity of steroidogenic enzymes.