Specific dopaminergic genetic variants influence impulsivity, cognitive deficit, and disease severity of Indian ADHD probands.

BACKGROUND: Impulsivity (Imp), being one of the cardinal symptoms of Attention Deficit Hyperactivity Disorder (ADHD), often leads to inappropriate responses to stimuli. Since the dopaminergic system is the primary target for pharmaceutical intervention in ADHD, we investigated the association between ADHD-related Imp and functional gene variants of the dopamine transporter (SLC6A3) and catechol-O-methyltransferase involved in dopamine clearance. METHODS AND RESULTS: Indo-Caucasoid families with ADHD probands (N = 217) were recruited based on the Diagnostic and Statistical Manual of Mental Disorders (DSM). Imp of the probands was assessed using the Domain Specific Imp Scale for Children and DSM. Peripheral blood was collected after obtaining informed written consent for participation, genomic DNA was isolated, and target sites were genotyped by DNA sequencing. The association of genetic variants with Imp was examined by the Quantitative trait analysis (QTA) and Analysis of variance (ANOVA). Post-Hoc analysis following QTA and ANOVA showed significant associations of rs2254408, rs2981359, and rs2239393 with different domains of Imp (P < 0.05). Various haplotypic combinations also showed statistically significant associations with Imp (P < 0.05). Multifactor dimensionality reduction models revealed strong effects of the variants on Imp. ADHD probands harboring the risk alleles exhibited a deficit in performance during cognitive assessment. Longitudinal follow-up revealed a significant association of rs2254408 with trait persistence. CONCLUSION: The present study indicates the influence of the studied genetic variants on ADHD-associated imp, executive deficit, and disease persistence. Thus, these variants may be helpful as predictors for the success of individual therapeutic sessions during cognitive training.

Synthesis and Characterization of Andrographolide Derivatives as Regulators of ßAPP Processing in Human Cells.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder, one of the main characteristics of which is the abnormal accumulation of amyloid peptide (Aß) in the brain. Whereas ß-secretase supports Aß formation along the amyloidogenic processing of the ß-amyloid precursor protein (ßAPP), α-secretase counterbalances this pathway by both preventing Aß production and triggering the release of the neuroprotective sAPPα metabolite. Therefore, stimulating α-secretase and/or inhibiting ß-secretase can be considered a promising anti-AD therapeutic track. In this context, we tested andrographolide, a labdane diterpene derived from the plant Andrographis paniculata, as well as 24 synthesized derivatives, for their ability to induce sAPPα production in cultured SH-SY5Y human neuroblastoma cells. Following several rounds of screening, we identified three hits that were subjected to full characterization. Interestingly, andrographolide (8,17-olefinic) and its close derivative 14α-(5',7'-dichloro-8'-quinolyloxy)-3,19-acetonylidene (compound 9) behave as moderate α-secretase activators, while 14α-(2'-methyl-5',7'-dichloro-8'-quinolyloxy)-8,9-olefinic compounds 31 (3,19-acetonylidene) and 37 (3,19-diol), whose two structures are quite similar although distant from that of andrographolide and 9, stand as ß-secretase inhibitors. Importantly, these results were confirmed in human HEK293 cells and these compounds do not trigger toxicity in either cell line. Altogether, these findings may represent an encouraging starting point for the future development of andrographolide-based compounds aimed at both activating α-secretase and inhibiting ß-secretase that could prove useful in our quest for the therapeutic treatment of AD.

Dopaminergic gene analysis indicates influence of inattention but not IQ in executive dysfunction of Indian ADHD probands.

Organizational inefficiency and inattention are speculated to be the reason for executive deficit (ED) of ADHD probands. Even with average IQ, probands often perform poorly due to higher inattention. Pharmacotherapy, cognitive behavioural therapy, and counselling provide only symptomatic relief. Several candidate genes showed involvement with ADHD; the most consistent are dopamine receptor 4 (DRD4) and solute carrier family 6 member 3 (SLC6A3). We analyzed association of rarely investigated DRD4 and SLC6A3 variants with ADHD core traits in Indo-Caucasoid probands. ED, inattention, organizational efficiency, and IQ were measured by Barkley Deficit in Executive Functioning-Child & Adolescent scale, DSM-IV-TR, Conners' Parent Rating Scale-revised, and WISC respectively. Target sites were analyzed by PCR, RFLP, and/or Sanger sequencing of genomic DNA. DRD4 variants mostly affected inattention while SLC6A3 variants showed association with IQ. Few DRD4 and SLC6A3 variants showed dichotomous association with IQ and inattention. DRD4 Exon3 VNTR >4R showed negative impact on all traits excepting IQ. Inattention showed correlation with attention span, organizational efficiency, and ED, while IQ failed to do so. We infer that IQ and attention could be differentially regulated by dopaminergic gene variants affecting functional efficiency in ADHD and the two traits should be considered together for providing better rehabilitation.

Parental age and developmental milestones: pilot study indicated a role in understanding ADHD severity in Indian probands.

BACKGROUND: In different ethnic groups, birth related factors have shown significant influence in the etiology of Attention deficit hyperactivity disorder (ADHD). Based on these interesting findings, we aimed to investigate association between different pre- and post natal variables and ADHD associated traits in Indian subjects. METHODS: ADHD Probands recruited based on the DSM-IV, were assessed by the Conner's Parent Rating Scale for behavioral problem (BPr), inattention (IA), hyperactivity (HA) and ADHD index (AI). Impulsivity (Imp) was assessed by the Tsukuyama scale. RESULTS: Higher paternal (Std ß = 0.23) and lower maternal (Std ß = 0.21) age showed significant association with Imp of the probands. Higher paternal age also revealed association with BPr (Std ß = 0.18). Age of onset was distinctly associated with AI (Std ß < 0.16) while developmental delay was negatively correlated with BPr, Imp, IA and birth weight (r < - 0.13); also confirmed by Posthoc-ANOVA (P < 0.05). CONCLUSION: We infer that parental age, developmental delay and birth related variables may have a cumulative effect on ADHD symptom severity.

Pilot study indicate role of preferentially transmitted monoamine oxidase gene variants in behavioral problems of male ADHD probands.

BACKGROUND: Attention deficit hyperactivity disorder (ADHD) is an etiologically complex childhood onset neurobehavioral disorder characterized by age-inappropriate inattention, hyperactivity, and impulsivity. Symptom severity varies widely and boys are diagnosed more frequently than girls. ADHD probands were reported to have abnormal transmissions of dopamine, serotonin, and/or noradrenaline. Monoamine oxidase A (MAOA) and B (MAOB), mitochondrial outer membrane bound two isoenzymes, mediate degradation of these neurotransmitters and thus regulating their circulating levels. Case-control analyses in different populations, including Indians, suggested involvement of MAOA and MAOB genes in the etiology of ADHD. Due to high heritability rate of ADHD, we tested familial transmission of MAOA and MAOB variants to ADHD probands in 190 nuclear families having ADHD probands from Indo-Caucasoid ethnicity. METHODS: Subjects were recruited following the Diagnostic and Statistical Manual of Mental Disorders-4th edition (DSM-IV). Appropriate scales were used for measuring the behavioral traits in probands. Genotyping was performed through PCR-based amplification of target sites followed by DNA-sequencing and/or gel-electrophoresis. Data obtained were analyzed by family based statistical methods. RESULTS: Out of 58 variants present in the analyzed sites only 15 were found to be polymorphic (30 bp-uVNTR, rs5906883, rs1465107, rs1465108, rs5905809, rs5906957, rs6323, rs1137070 from MAOA and rs4824562, rs56220155, rs2283728, rs2283727, rs3027441, rs6324, rs3027440 from MAOB). Statistically significant maternal transmission of alleles to male probands was observed for MAOA rs5905809 'G' (p = 0.04), rs5906957 'A' (p = 0.04), rs6323 'G' (p = 0.0001) and MAOB rs56220155 'A' (p = 0.002), rs2283728 'C' (p = 0.0008), rs2283727 'C' (p = 0.0008), rs3027441 'T' (p = 0.003), rs6324 'C' (p = 0.003), rs3027440 'T' (p = 0.0002). Significantly preferential maternal transmissions of different haplotype combinations to male probands were also noticed (p < 0.05), while female probands did not reveal such transmission bias. Behavioral traits of male probands exhibited significant association with gene variants. Age of the mother at pregnancy also revealed association with risk variants of male probands. CONCLUSIONS: It may be inferred that the MAOA and MAOB variants may contribute to the etiology of ADHD in the Indo-Caucasoid population and could be responsible for higher occurrence of ADHD in the boys.

Monoamine oxidase B gene variants associated with attention deficit hyperactivity disorder in the Indo-Caucasoid population from West Bengal.

BACKGROUND: Attention deficit hyperactivity disorder (ADHD) is characterized by symptoms of inattention, excessive motor activity and impulsivity detected mostly during childhood. These traits are known to be controlled by monoamine neurotransmitters, chiefly dopamine, serotonin and norepinephrine. Monoamine oxidase A (MAOA) and B (MAOB), two isoenzymes bound to the outer membrane of mitochondria, are involved in the degradation of monoamines and were explored for association with ADHD in different ethnic groups. In the present study, few exonic as well as intronic MAOB variants were analyzed in ADHD probands (N = 150) and ethnically matched controls (N = 150) recruited following the Diagnostic and Statistical Manual for Mental Disorders-4(th) edition (DSM-IV). Appropriate scales were used for measuring the behavioural attributes. Gene variants were analyzed by amplification of target sites followed by DNA sequencing and data obtained were analyzed by population based statistical methods. RESULTS: Out of 34 variants present in the analyzed sites, only seven functional variants, rs4824562, rs56220155, rs2283728, rs2283727, rs3027441, rs6324 and rs3027440, were found to be polymorphic. rs2283728 'C' (P = 3.45e-006) and rs3027440 'T' (P = 0.02) alleles showed higher frequencies in ADHD probands as compared to controls. rs56220155 'A' (P = 0.04) allele and 'GA' (P = 0.04) genotype showed higher frequencies in the male and female ADHD probands respectively as compared to sex-matched controls. Analysis of pairwise linkage disequilibrium revealed striking differences between probands and controls. Haplotype analysis revealed significantly higher occurrence of different haplotypes in the ADHD probands while some haplotypes were detected in the controls only. Higher scores for conduct problems were found to be associated with rs56220155 'A' (P = 0.05) allele in the male ADHD probands. Multifactor dimensionality reduction analysis showed independent as well as interactive effects of polymorphic variants which were more robust in the male probands. CONCLUSIONS: Since all the polymorphic variants analyzed were functional, it may be inferred that MAOB gene variants are contributing to the etiology of ADHD in the Indo-Caucasoid population from eastern India which merits further in depth analysis.

Potential contribution of dopaminergic gene variants in ADHD core traits and co-morbidity: a study on eastern Indian probands.

Association of dopaminergic genes, mainly receptors and transporters, with Attention Deficit Hyperactivity Disorder (ADHD) has been investigated throughout the world due to the importance of dopamine (DA) in various physiological functions including attention, cognition and motor activity, traits. However, till date, etiology of ADHD remains unknown. We explored association of functional variants in the DA receptor 2 (rs1799732 and rs6278), receptor 4 (exon 3 VNTR and rs914655), and transporter (rs28363170 and rs3836790) with hyperactivity, cognitive deficit, and co-morbid disorders in eastern Indian probands. Diagnostic and Statistical Manual for Mental Disorders-IV was followed for recruitment of nuclear families with ADHD probands (N = 160) and ethnically matched controls (N = 160). Cognitive deficit and hyperactive traits were measured using Conner's parents/teachers rating scale. Peripheral blood was collected after obtaining informed written consent and used for genomic DNA isolation. Genetic polymorphisms were analyzed by PCR-based methods followed by population- as well as family-based statistical analyses. Association between genotypes and cognitive/hyperactivity traits and co-morbidities was analyzed by the Multifactor dimensionality reduction (MDR) software. Case-control analysis showed statistically significant difference for rs6278 and rs28363170 (P = 0.004 and 1.332e-007 respectively) while family-based analysis exhibited preferential paternal transmission of rs28363170 '9R' allele (P = 0.04). MDR analyses revealed independent effects of rs1799732, rs6278, rs914655, and rs3836790 in ADHD. Significant independent effects of different sites on cognitive/hyperactivity traits and co-morbid disorders were also noticed. It can be summarized from the present investigation that these gene variants may influence cognitive/hyperactive traits, thereby affecting the disease etiology and associated co-morbid features.

BACE1-dependent metabolism of neuregulin 1: Bridging the gap in explaining the occurrence of schizophrenia-like symptoms in Alzheimer's disease with psychosis?

Alzheimer's disease is a neurodegenerative disease mainly characterized by cortico-neuronal atrophy, impaired memory and other cognitive declines. On the other hand, schizophrenia is a neuro-developmental disorder with an overtly active central nervous system pruning system resulting into abrupt connections with common symptoms including disorganised thoughts, hallucination and delusion. Nevertheless, the fronto-temporal anomaly presents itself as a common denominator for the two pathologies. There is even a strong presumption of increased risk of developing co-morbid dementia for schizophrenic individuals and psychosis for Alzheimer's disease patients, overall leading to a further deteriorated quality of life. However, convincing proofs of how these two disorders, although very distant from each other when considering their aetiology, develop coexisting symptoms is yet to be resolved. At the molecular level, the two primarily neuronal proteins ß-amyloid precursor protein and neuregulin 1 have been considered in this relevant context, although the conclusions are for the moment only hypotheses. In order to propose a model for explaining the psychotic schizophrenia-like symptoms that sometimes accompany AD-associated dementia, this review projects out on the similar sensitivity shared by these two proteins regarding their metabolism by the ß-site APP cleaving enzyme 1.

Post-treatment symptomatic improvement of the eastern Indian ADHD probands is influenced by CYP2D6 genetic variations.

OBJECTIVES: Symptomatic remediation from attention deficit hyperactivity disorder (ADHD)-associated traits is achieved by treatment with methylphenidate (MPH)/atomoxetine (ATX). We have analyzed the association of functional CYP2D6 variations, rs1065852, rs3892097, rs1135840, and rs1058164, with ADHD in the Indian subjects. METHODS: Subjects were recruited following the Diagnostic and Statistical Manual for Mental Disorders. Trait scores were obtained from the Conner's Parents Rating Scale-Revised. After obtaining informed consent, blood was collected for DNA isolation, and genotyping was performed by PCR or TaqMan-based methods. Probands were treated with MPH or ATX based on age, symptoms, and drug availability. Treatment outcome was assessed using a structured questionnaire. Data obtained was analyzed to identify the association of CYP2D6 variations and the SLC6A3 rs28363170 with the treatment outcome. RESULTS: The frequency of rs1135840 "G" and rs1065852 "G" was higher in the male ADHD probands. Bias in parental transmission (p=0.007) and association with higher trait scores were observed for rs1065852 "A". Independent influence of rs1065852 on ADHD was also observed. Probands carrying rs1065852 'GG', rs1135840 'CG', and rs28363170 10R exhibited significant symptomatic improvement with MPH, while probands with rs1135840 'CC' and rs28363170 9R showed improvement after ATX treatment. CONCLUSIONS: ADHD probands having specific CYP2D6 genetic variations respond differentially to pharmaceutical intervention.

Cdk5-p25 as a key element linking amyloid and tau pathologies in Alzheimer's disease: Mechanisms and possible therapeutic interventions.

Despite the fact that the small atypical serine/threonine cyclin-dependent kinase 5 (Cdk5) is expressed in a number of tissues, its activity is restricted to the central nervous system due to the neuron-only localization of its activators p35 and p39. Although its importance for the proper development and function of the brain and its role as a switch between neuronal survival and death are unmistakable and unquestionable, Cdk5 is nevertheless increasingly emerging, as supported by a large number of publications on the subject, as a therapeutic target of choice in the fight against Alzheimer's disease. Thus, its aberrant over activation via the calpain-dependent conversion of p35 into p25 is observed during the pathogenesis of the disease where it leads to the hyperphosphorylation of the ß-amyloid precursor protein and tau. The present review highlights the pivotal roles of the hyperactive Cdk5-p25 complex activity in contributing to the development of Alzheimer's disease pathogenesis, with a particular emphasis on the linking function between Aß and tau that this kinase fulfils and on the fact that Cdk5-p25 is part of a deleterious feed forward loop giving rise to a molecular machinery runaway leading to AD pathogenesis. Additionally, we discuss the advances and challenges related to the possible strategies aimed at specifically inhibiting Cdk5-p25 activity and which could lead to promising anti-AD therapeutics.

Phenanthroline impairs ßAPP processing and expression, increases p53 protein levels and induces cell cycle arrest in human neuroblastoma cells.

Mis-functional ßAPP processing is deemed to be the major phenomenon resulting in increased neuronal cell death, impaired neurogenesis and the loss of synapses, which eventually manifest as the complex symptoms of Alzheimer's disease. Despite of several milestones having been achieved in the field of drug development, the stigma of the disorder as an incurable disease still remains. Some ADAM proteases mediate the physiological non-amyloidogenic α-secretase processing of ßAPP that generates neuroprotective sAPPα production. Earlier studies have also pointed out the role of p53 in Alzheimer's disease neuropathology, although a direct link with metalloprotease activities remains to be established. In this study, we explored the consequences of α-secretase inhibition on p53 status in cultured human neuroblastoma SH-SY5Y cells by means of specific inhibitors of ADAM10 and ADAM17 and the metal chelator and general metalloprotease inhibitor phenanthroline. We establish that, beyond the ability of all inhibitors to affect sAPPα production to varying degrees, phenanthroline specifically and dose-dependently lessened ßAPP expression, a phenomenon that correlated with a strong increase in p53 protein levels and a concomitant decrease of the p53-degrading calpain protease. Furthermore, treatment of cells at concentrations of phenanthroline similar to those inducing increased levels of p53 induced cell cycle arrest leading to apoptosis. Altogether, our results identify new roles of phenanthroline in perturbing ßAPP, p53 and calpain biology, and suggest that the use of this compound and its derivatives as antimicrobial and anti-cancer therapies might trigger Alzheimer's disease pathogenesis.

Folate System Gene Variant rs1801394 66A>G may have a Causal Role in Down Syndrome in the Eastern Indian Population.

Down syndrome (DS) is associated with trisomy of the 21st chromosome in more than 95% cases. The extra chromosome mostly derives due to abnormal chromosomal segregation, i.e. non-disjunction, during meiosis. Earlier reports showed that abnormal folate metabolism can lead to DNA hypomethylation and abnormal chromosomal segregation. We analyzed three functional folate gene variants, namely 5-methyltetrahydrofolate-homocysteine methyltransferase rs1805087, 5-methyltetrahydrofolate-homocysteine methyltransferase reductase rs1801394, and reduced folate carrier 1 rs1051266, for contribution in the etiology of DS. Ethnically matched subjects including DS probands (N=183), their parents (N=273), and controls (N=286) were recruited after obtaining informed written consent for participation. Karyotype analysis confirmed trisomy 21 in DS patients recruited. Genomic DNA, purified from peripheral blood leukocytes was used for genotyping of the target sites by PCR based methods, and data obtained was subjected to population- as well as family-based association analysis. Frequency of rs1801394 'G' allele and 'GG' genotype was higher in DS probands (P < 0.0001). Statistically significant higher occurrence of the 'G' allele in parents of DS probands (P < 0.0001) and maternal bias in transmission of the "G" allele was also noticed (P < 0.0001). Genetic model analysis demonstrated rs1801394 "G" as a risk allele under both dominant and recessive models. DS probands also showed higher occurrence of rs1051266 "G" (P = 0.05). Quantitative trait analysis revealed significant negative influence of rs1805087 "A" on birth weight. Screening for rs1801394 "G" could be useful in monitoring the risk of DS, at least in the studied population.

Methamphetamine regulates ßAPP processing in human neuroblastoma cells.

Methamphetamine is a potent and highly addictive psychostimulant whose abuse has turned out to be a global health hazard. The multitudinous effects it exerts at the cellular level induces neurotoxic responses in the human brain, ultimately leading to neurocognitive disorders. Strikingly, brain changes, tissue damage and neuropsychological symptoms due to Meth exposure compels and necessitates to link the probability of risk of developing premature Alzheimer's disease, a progressive neurodegenerative disorder characterized by amyloid plaques composed of amyloid-ß peptides and clinical dementia. These peptides are derived from sequential cleavages of the ß-amyloid precursor protein by ß- and γ-secretases. Previous studies reveals evidence for both positive and negative effects of Meth pertaining to cognitive functioning based on the dosage paradigm and duration of exposure revealing a beneficial psychotropic profile under some conditions and deleterious cognitive deficits under some others. In this context, we proposed to examine the effect of Meth on ßAPP metabolism and ßAPP-cleaving secretases in the human neuroblastoma SH-SY5Y cell line. Our results showed that Meth dose-dependently increases BACE1 expression and catalytic activity, while its effect on the α-cleavage of ßAPP and on the expression and catalytic activity of the main α-secretase ADAM10 display a bell-curve shape. To our knowledge, the present study is the first to demonstrate that Meth can control ßAPP-cleaving secretases. Moreover, we propose from these findings that the deleterious effect of Meth on cognitive decline might be an outcome of high dosage paradigm whereas acute and short-term drug use which stimulated sAPPα might produce improvements in cognition in disorders such as AD.

Cyclin-dependent Kinase 5: Novel role of gene variants identified in ADHD.

Cortical neuronal migration and formation of filamentous actin cytoskeleton, needed for development, normal cell growth and differentiation, are regulated by the cyclin-dependent kinase 5 (Cdk5). Attention deficit hyperactivity disorder (ADHD) is associated with delayed maturation of the brain and hence we hypothesized that cdk5 may have a role in ADHD. Eight functional CDK5 gene variants were analyzed in 848 Indo-Caucasoid individuals including 217 families with ADHD probands and 250 healthy volunteers. Only three variants, rs2069454, rs2069456 and rs2069459, predicted to affect transcription, were found to be bimorphic. Significant difference in rs2069456 "AC" genotype frequency was noticed in the probands, more specifically in the males. Family based analysis revealed over transmission of rs2069454 "C" and rs2069456 "A" to the probands. Quantitative trait analysis exhibited association of haplotypes with inattention, domain specific impulsivity, and behavioral problem, though no significant contribution was noticed on the age of onset of ADHD. Gene variants also showed significant association with cognitive function and co-morbidity. Probands having rs2069459 "TT" showed betterment during follow up. It may be inferred from this pilot study that CDK5 may affect ADHD etiology, possibly by attenuating synaptic neurotransmission and could be a useful target for therapeutic intervention.

The Dopamine Receptor D5 May Influence Age of Onset: An Exploratory Study on Indo-Caucasoid ADHD Subjects.

The objective was to investigate contribution of the dopamine receptor 5 (DRD5) gene variants in the symptoms of attention-deficit/hyperactivity disorder (ADHD) probands since brain regions identified to be affected in these group of patients have higher expression of the DRD5 receptor. Out of 22 exonic variants, 19 were monomorphic in the Indo-Caucasoid individuals. rs6283 "C" and rs113828117 "A" exhibited significant higher occurrence in families with ADHD probands. Several haplotypes showed biased occurrence in the probands. Early and late onset groups exhibited significantly different genotypic frequencies. A new G>A substitution was observed in the control samples only. The late onset group exhibited higher scores for hyperactivity as compared to the early onset group. The authors infer that the age of onset of ADHD may at least partially be affected by DRD5 variants warranting further investigation on the role of DRD5 in the disease etiology.