Tiziana Pascucci

Altered calcium homeostasis in autism-spectrum disorders: evidence from biochemical and genetic studies of the mitochondrial aspartate/glutamate carrier AGC1

Autism is a severe developmental disorder, whose pathogenetic underpinnings are still largely unknown. Temporocortical gray matter from six matched patient–control pairs was used to perform post-mortem biochemical and genetic studies of the mitochondrial aspartate/glutamate carrier (AGC), which participates in the aspartate/malate reduced nicotinamide adenine dinucleotide shuttle and is physiologically activated by calcium (Ca2+). AGC transport rates were significantly higher in tissue homogenates from all six patients, including those with no history of seizures and with normal electroencephalograms prior to death. This increase was consistently blunted by the Ca2+ chelator ethylene glycol tetraacetic acid; neocortical Ca2+ levels were significantly higher in all six patients; no difference in AGC transport rates was found in isolated mitochondria from patients and controls following removal of the Ca2+-containing postmitochondrial supernatant. Expression of AGC1, the predominant AGC isoform in brain, and cytochrome c oxidase activity were both increased in autistic patients, indicating an activation of mitochondrial metabolism. Furthermore, oxidized mitochondrial proteins were markedly increased in four of the six patients. Variants of the AGC1-encoding SLC25A12 gene were neither correlated with AGC activation nor associated with autism-spectrum disorders in 309 simplex and 17 multiplex families, whereas some unaffected siblings may carry a protective gene variant. Therefore, excessive Ca2+ levels are responsible for boosting AGC activity, mitochondrial metabolism and, to a more variable degree, oxidative stress in autistic brains. AGC and altered Ca2+ homeostasis play a key interactive role in the cascade of signaling events leading to autism: their modulation could provide new preventive and therapeutic strategies.

Identifying Molecular Substrates in a Mouse Model of the Serotonin Transporter × Environment Risk Factor for Anxiety and Depression

BACKGROUND A polymorphism in the serotonin transporter (5-HTT) gene modulates the association between adverse early experiences and risk for major depression in adulthood. Although human imaging studies have begun to elucidate the neural circuits involved in the 5-HTT x environment risk factor, a molecular understanding of this phenomenon is lacking. Such an understanding might help to identify novel targets for the diagnosis and therapy of mood disorders. To address this need, we developed a gene-environment screening paradigm in the mouse. METHODS We established a mouse model in which a heterozygous null mutation in 5-HTT moderates the effects of poor maternal care on adult anxiety and depression-related behavior. Biochemical analysis of brains from these animals was performed to identify molecular substrates of the gene, environment, and gene x environment effects. RESULTS Mice experiencing low maternal care showed deficient gamma-aminobutyric acid-A receptor binding in the amygdala and 5-HTT heterozygous null mice showed decreased serotonin turnover in hippocampus and striatum. Strikingly, levels of brain-derived neurotrophic factor (BDNF) messenger RNA in hippocampus were elevated exclusively in 5-HTT heterozygous null mice experiencing poor maternal care, suggesting that developmental programming of hippocampal circuits might underlie the 5-HTT x environment risk factor. CONCLUSIONS These findings demonstrate that serotonin plays a similar role in modifying the long-term behavioral effects of rearing environment in diverse mammalian species and identifies BDNF as a molecular substrate of this risk factor.

TLQP-21, a VGF-derived peptide, increases energy expenditure and prevents the early phase of diet-induced obesity

The vgf gene has been identified as an energy homeostasis regulator. Vgf encodes a 617-aa precursor protein that is processed to yield an incompletely characterized panel of neuropeptides. Until now, it was an unproved assumption that VGF-derived peptides could regulate metabolism. Here, a VGF peptide designated TLQP-21 was identified in rat brain extracts by means of immunoprecipitation, microcapillary liquid chromatography–tandem MS, and database searching algorithms. Chronic intracerebroventricular (i.c.v.) injection of TLQP-21 (15 μg/day for 14 days) increased resting energy expenditure (EE) and rectal temperature in mice. These effects were paralleled by increased epinephrine and up-regulation of brown adipose tissue β2-AR (β2 adrenergic receptor) and white adipose tissue (WAT) PPAR-δ (peroxisome proliferator-activated receptor δ), β3-AR, and UCP1 (uncoupling protein 1) mRNAs and were independent of locomotor activity and thyroid hormones. Hypothalamic gene expression of orexigenic and anorexigenic neuropeptides was unchanged. Furthermore, in mice that were fed a high-fat diet for 14 days, TLQP-21 prevented the increase in body and WAT weight as well as hormonal changes that are associated with a high-fat regimen. Biochemical and molecular analyses suggest that TLQP-21 exerts its effects by stimulating autonomic activation of adrenal medulla and adipose tissues. In conclusion, we present here the identification in the CNS of a previously uncharacterized VGF-derived peptide and prove that its chronic i.c.v. infusion effected an increase in EE and limited the early phase of diet-induced obesity.

Paraoxonase gene variants are associated with autism in North America, but not in Italy: possible regional specificity in gene-environment interactions

Organophosphates (OPs) are routinely used as pesticides in agriculture and as insecticides within the household. Our prior work on Reelin and APOE delineated a gene–environment interactive model of autism pathogenesis, whereby genetically vulnerable individuals prenatally exposed to OPs during critical periods in neurodevelopment could undergo altered neuronal migration, resulting in an autistic syndrome. Since household use of OPs is far greater in the USA than in Italy, this model was predicted to hold validity in North America, but not in Europe. Here, we indirectly test this hypothesis by assessing linkage/association between autism and variants of the paraoxonase gene (PON1) encoding paraoxonase, the enzyme responsible for OP detoxification. Three functional single nucleotide polymorphisms, PON1 C−108T, L55M, and Q192R, were assessed in 177 Italian and 107 Caucasian-American complete trios with primary autistic probands. As predicted, Caucasian-American and not Italian families display a significant association between autism and PON1 variants less active in vitro on the OP diazinon (R192), according to case–control contrasts (Q192R: χ2=6.33, 1 df, P<0.025), transmission/disequilibrium tests (Q192R: TDT χ2=5.26, 1 df, P<0.025), family-based association tests (Q192R and L55M: FBAT Z=2.291 and 2.435 respectively, P<0.025), and haplotype-based association tests (L55/R192: HBAT Z=2.430, P<0.025). These results are consistent with our model and provide further support for the hypothesis that concurrent genetic vulnerability and environmental OP exposure may possibly contribute to autism pathogenesis in a sizable subgroup of North American individuals.

Activation of TRPV1 in the VTA excites dopaminergic neurons and increases chemical- and noxious-induced dopamine release in the nucleus accumbens.

Dopamine (DA)-containing neurons of the ventral tegmental area (VTA) provide dopaminergic input to the nucleus accumbens and to the prefrontal cortex within the mesolimbic pathway. In the present study, we combined electrophysiological recordings and microdialysis techniques to investigate the function of transient receptor potential vanilloid 1 (TRPV1) channel in the VTA. In brain slices, application of the TRPV1 receptor agonist capsaicin increased the firing rate of rat dopamine neurons and in a proportion of tested cells (44%) it also induced a bursting behavior. The effects of capsaicin were concentration dependent. The increase in neuronal firing was dependent on enhanced glutamatergic transmission since it was blocked by the superfusion of the ionotropic glutamate antagonists, CNQX and AP5. Interestingly, microinjection of capsaicin into the VTA and noxious tail stimulation transiently enhanced dopamine release into the nucleus accumbens. Both the in vitro and in vivo effects were mediated by TRPV1 activation in the VTA since they were reduced by co-perfusion of the selective TRPV1 receptor antagonist iodoresineferatoxin. Our data suggest a novel role for TRPV1 channels in the mesencephalon of rat, namely activation of the DA system following a peripheral noxious stimulation.

Clinical, morphological, and biochemical correlates of head circumference in autism

BACKGROUND Head growth rates are often accelerated in autism. This study is aimed at defining the clinical, morphological, and biochemical correlates of head circumference in autistic patients. METHODS Fronto-occipital head circumference was measured in 241 nonsyndromic autistic patients, 3 to 16 years old, diagnosed according to DSM-IV criteria. We assessed 1) clinical parameters using the Autism Diagnostic Observation Schedule, Autism Diagnostic Interview-Revised, Vineland Adaptive Behavioral Scales, intelligence quotient measures, and an ad hoc clinical history questionnaire; 2) height and weight; 3) serotonin (5-HT) blood levels and peptiduria. RESULTS The distribution of cranial circumference is significantly skewed toward larger head sizes (p < .00001). Macrocephaly (i.e., head circumference >97th percentile) is generally part of a broader macrosomic endophenotype, characterized by highly significant correlations between head circumference, weight, and height (p < .001). A head circumference >75th percentile is associated with more impaired adaptive behaviors and with less impairment in IQ measures and motor and verbal language development. Surprisingly, larger head sizes are significantly associated with a positive history of allergic/immune disorders both in the patient and in his/her first-degree relatives. CONCLUSIONS Our study demonstrates the existence of a macrosomic endophenotype in autism and points toward pathogenetic links with immune dysfunctions that we speculate either lead to or are associated with increased cell cycle progression and/or decreased apoptosis.

Association between the HOXA1 A218G polymorphism and increased head circumference in patients with autism

BACKGROUND The HOXA1 gene plays a major role in brainstem and cranial morphogenesis. The G allele of the HOXA1 A218G polymorphism has been previously found associated with autism. METHODS We performed case-control and family-based association analyses, contrasting 127 autistic patients with 174 ethnically matched controls, and assessing for allelic transmission disequilibrium in 189 complete trios. RESULTS A, and not G, alleles were associated with autism using both case-control (chi(2) = 8.96 and 5.71, 1 df, p <.005 and <.025 for genotypes and alleles, respectively), and family-based (transmission/disequilibrium test chi(2) = 8.80, 1 df, p <.005) association analyses. The head circumference of 31 patients carrying one or two copies of the G allele displayed significantly larger median values (95.0th vs. 82.5th percentile, p <.05) and dramatically reduced interindividual variability (p <.0001), compared with 166 patients carrying the A/A genotype. CONCLUSIONS The HOXA1 A218G polymorphism explains approximately 5% of the variance in the head circumference of autistic patients and represents to our knowledge the first known gene variant providing sizable contributions to cranial morphology. The disease specificity of this finding is currently being investigated. Nonreplications in genetic linkage/association studies could partly stem from the dyshomogeneous distribution of an endophenotype morphologically defined by cranial circumference.

Principal pathogenetic components and biological endophenotypes in autism spectrum disorders

Autism is a complex neurodevelopmental disorder, likely encompassing multiple pathogenetic components. The aim of this study is to begin identifying at least some of these components and to assess their association with biological endophenotypes. To address this issue, we recruited 245 Italian patients with idiopathic autism spectrum disorders and their first‐degree relatives. Using a stepwise approach, patient and family history variables were analyzed using principal component analysis (“exploratory phase”), followed by intra‐ and inter‐component cross‐correlation analyses (“follow‐up phase”), and by testing for association between each component and biological endophenotypes, namely head circumference, serotonin blood levels, and global urinary peptide excretion rates (“biological correlation phase”). Four independent components were identified, namely “circadian & sensory dysfunction,” “immune dysfunction,” “neurodevelopmental delay,” and “stereotypic behavior,” together representing 74.5% of phenotypic variance in our sample. Marker variables in the latter three components are positively associated with macrocephaly, global peptiduria, and serotonin blood levels, respectively. These four components point toward at least four processes associated with autism, namely (I) a disruption of the circadian cycle associated with behavioral and sensory abnormalities, (II) dysreactive immune processes, surprisingly linked both to prenatal obstetric complications and to excessive postnatal body growth rates, (III) a generalized developmental delay, and (IV) an abnormal neural circuitry underlying stereotypies and early social behaviors.

Increased vulnerability to psychosocial stress in heterozygous serotonin transporter knockout mice

SUMMARY Epidemiological evidence links exposure to stressful life events with increased risk for mental illness. However, there is significant individual variability in vulnerability to environmental risk factors, and genetic variation is thought to play a major role in determining who will become ill. Several studies have shown, for example, that individuals carrying the S (short) allele of the serotonin transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR) have an increased risk for major depression following exposure to stress in adulthood. Identifying the molecular mechanisms underlying this gene-by-environment risk factor could help our understanding of the individual differences in resilience to stress. Here, we present a mouse model of the 5-HTT-by-stress risk factor. Wild-type and heterozygous 5-HTT knockout male mice were subjected to three weeks of chronic psychosocial stress. The 5-HTT genotype did not affect the physiological consequences of stress as measured by changes in body temperature, body weight gain and plasma corticosterone. However, when compared with wild-type littermates, heterozygous 5-HTT knockout mice experiencing high levels of stressful life events showed significantly depressed locomotor activity and increased social avoidance toward an unfamiliar male in a novel environment. Heterozygous 5-HTT knockout mice exposed to high stress also showed significantly lower levels of serotonin turnover than wild-type littermates, selectively in the frontal cortex, which is a structure that is known to control fear and avoidance responses, and that is implicated in susceptibility to depression. These data may serve as a useful animal model for better understanding the increased vulnerability to stress reported in individuals carrying the 5-HTTLPR S allele, and suggest that social avoidance represents a behavioral endophenotype of the interaction between 5-HTT and stress.

Mechanisms underlying the impairment of hippocampal long-term potentiation and memory in experimental Parkinson's disease.

Although patients with Parkinsons disease show impairments in cognitive performance even at the early stage of the disease, the synaptic mechanisms underlying cognitive impairment in this pathology are unknown. Hippocampal long-term potentiation represents the major experimental model for the synaptic changes underlying learning and memory and is controlled by endogenous dopamine. We found that hippocampal long-term potentiation is altered in both a neurotoxic and transgenic model of Parkinsons disease and this plastic alteration is associated with an impaired dopaminergic transmission and a decrease of NR2A/NR2B subunit ratio in synaptic N-methyl-d-aspartic acid receptors. Deficits in hippocampal-dependent learning were also found in hemiparkinsonian and mutant animals. Interestingly, the dopamine precursor l-DOPA was able to restore hippocampal synaptic potentiation via D1/D5 receptors and to ameliorate the cognitive deficit in parkinsonian animals suggesting that dopamine-dependent impairment of hippocampal long-term potentiation may contribute to cognitive deficits in patients with Parkinsons disease.