1 Nature Medicine 2011 Vol: 17(5):566-572. DOI: 10.1038/nm.2330

GIT1 is associated with ADHD in humans and ADHD-like behaviors in mice

Attention deficit hyperactivity disorder (ADHD) is a psychiatric disorder that affects ~5% of school-aged children; however, the mechanisms underlying ADHD remain largely unclear. Here we report a previously unidentified association between G protein–coupled receptor kinase–interacting protein-1 (GIT1) and ADHD in humans. An intronic single-nucleotide polymorphism in GIT1, the minor allele of which causes reduced GIT1 expression, shows a strong association with ADHD susceptibility in humans. Git1-deficient mice show ADHD-like phenotypes, with traits including hyperactivity, enhanced electroencephalogram theta rhythms and impaired learning and memory. Hyperactivity in Git1−/− mice is reversed by amphetamine and methylphenidate, psychostimulants commonly used to treat ADHD. In addition, amphetamine normalizes enhanced theta rhythms and impaired memory. GIT1 deficiency in mice leads to decreases in ras-related C3 botulinum toxin substrate-1 (RAC1) signaling and inhibitory presynaptic input; furthermore, it shifts the neuronal excitation-inhibition balance in postsynaptic neurons toward excitation. Our study identifies a previously unknown involvement of GIT1 in human ADHD and shows that GIT1 deficiency in mice causes psychostimulant-responsive ADHD-like phenotypes.

Mentions
Figures
Figure 1: Hyperactivity and impaired memory in Git1−/− mice are normalized by amphetamine treatment. (a) Genotyping of Git1−/− mice by PCR (top), and undetectable GIT1 proteins in whole brain homogenates from Git1−/− mice (bottom; 6–10 weeks). KO, knockout; WT, wild type. α-tubulin was used as a control. (b,c) Locomotor activity of WT and Git1−/− mice in an open field. n = 15 (WT), n = 14 (KO); see also Supplementary Table 4. **P < 0.01, ***P < 0.001, NS, not significant; Student's t test. The three asterisks in the top right corner of the panel indicate a significant difference between two genotypes over time, as calculated by repeated-measures analysis of variance (ANOVA) (see Supplementary Table 5 for details of statistical results). (d) Novel-object recognition in WT and Git1−/− mice. n = 11 (WT), n = 12 (KO). *P < 0.05; Student's t test. (e–g) Spatial learning and memory in WT and Git1−/− mice, shown by escape latencies in the Morris water maze (e), target quadrant occupancy (f) and numbers of platform crossings (g). n = 16 (WT), n = 19 (KO). *P < 0.05, **P < 0.01, ***P < 0.001; Student's t test and repeated-measures ANOVA. (h,i) Effects of amphetamine (amph) and saline (sal; control) on locomotor activities of WT and Git1−/− mice in an open field. The results in h were quantified in i over a 10–20 min period. n = 9 (sal), n = 6 (amph) for WT; n = 7 (sal), n = 8 (amph) for KO. **P < 0.01, ***P < 0.001, one-way ANOVA. Details of statistical results for h (Student's t test) are described in Supplementary Table 5. (j) Effect of amphetamine (amph) and saline (sal) on novel-object recognition behavior of Git1−/− mice. Saline-treated WT mice were used for comparison. n = 7 (WT sal, KO amph, KO sal). *P < 0.05, **P < 0.01; one-way ANOVA. Error bars indicate means ± s.e.m. Figure 2: Enhanced theta rhythms in the frontal cortex of Git1−/− mice are reduced by amphetamine. (a–c) Theta EEG rhythms in the frontal cortex of WT and Git1−/− mice, as shown by representative traces and spectrogram (a), theta power (3–10 Hz; 10–20 min) (b) and number of theta events (c), in which a theta event is defined as a group of theta oscillations independent from others. A typical theta range in mice (3–10 Hz) is slightly different from that in humans (4–8 Hz). n = 5 (WT, KO). *P < 0.05, ***P < 0.001; Student's t test and repeated-measures ANOVA. (d–f) Effects of amphetamine and saline on enhanced theta rhythms in Git1−/− mice. n = 7 (KO sal), n = 6 (KO amph). *P < 0.05, ***P < 0.001; Student's t test and repeated-measures ANOVA. Error bars indicate means ± s.e.m. Figure 3: Suppressed GIT1-PIX-RAC1-PAK signaling in the Git1−/− brain. (a) Amounts of PIX proteins (α-PIX and β-PIX) and activity of RAC1 (a downstream effector of PIX) in the WT and Git1−/− brain, as determined by immunoblotting analysis of whole brain homogenates (for PIX) and pull-down analysis of GTP-bound (active) RAC1 with GST–p21 binding domain (GST-PBD). (b) Amounts of PAK (PAK1 and PAK3) and phosphorylated PAK (pPAK1 and pPAK3) proteins shown by immunoblotting. (c) Amounts of GIT1-interacting proteins (FAK, MEK, PLC-γ, and liprin-α1) and other synaptic proteins in the WT and Git1−/− brain. n = 6 (WT, KO) for immunoblotting analysis, n = 3 (WT, KO) for pull-down analysis. *P < 0.05, ***P < 0.001; Student's t test. FAK, focal adhesion kinase; MEK, mitogen-activated protein kinase kinase; PLC-γ, phospholipase C-γ; ERK1/2, extracellular-regulated kinase 1 and 2; pERK1/2, phosphorylated ERK1/2; GRIP1, glutamate receptor-interacting protein 1; ERC2, ELKS/Rab6IP2/CAST2; PSD-93/PSD-95, postsynaptic density 93/95; CASK, calcium/calmodulin-dependent serine protein kinase; NR1, NR2A and NR2B, subunits of NMDA glutamate receptors (also known as GluN1, GluN2A and GluN2B); GluR1 and GluR2, subunits of AMPA glutamate receptors (also known as GluA1 and GluA2). α-tubulin and β-actin were used as controls. Error bars indicate means ± s.e.m. Figure 4: Reduced inhibitory transmission and elevated excitatory transmission at Git1−/− synapses. (a) Amplitude and frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in WT and Git1−/− hippocampal CA1 pyramidal neurons. n = 17 cells from three mice for WT and n = 15 cells from three mice for KO. (b) Frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs) in WT and Git1−/− CA1 pyramidal neurons. n = 22 cells from three mice (WT), n = 23 cells from three mice (KO). **P < 0.01; Student's t test. (c) Amounts of charge transfer for mEPSCs and mIPSCs in WT and Git1−/− mice. n = 17 cells from three mice for WT, n = 15 cells from three mice for KO (mEPSCs), n = 22 cells from three mice for WT, n = 23 cells from three mice for KO (mIPSCs). (d) Excitatory transmission at WT and Git1−/− SC-CA1 synapses. The initial slopes of field EPSPs were plotted against fiber volley amplitudes. n = 41 slices from ten mice (WT), n = 27 slices from nine mice (KO). ***P < 0.001; Student's t test. (e) Presynaptic release probabilities at WT and Git1−/− SC-CA1 synapses, as measured by paired-pulse facilitation ratios (second fEPSP/first fEPSP) at different interstimulus intervals. n = 23 slices from eight mice (WT), n = 16 slices from six mice (KO). Error bars indicate means ± s.e.m. Figure 5: Reduced presynaptic input at Git1−/− inhibitory synapses. (a,b) Reduced amounts of inhibitory presynaptic proteins (GAD67 and vGAT) in the CA1 region of the Git1−/− hippocampus, which contrast to normal amounts of the inhibitory postsynaptic protein gephyrin and the presynaptic active zone protein bassoon, which is present at both excitatory and inhibitory synapses. Signals from the somas and processes of Git1−/− slices were normalized to those from WT mice. n = 3 slices from three mice (WT, KO). *P < 0.05; Student's t test. (c,d) Reduced amounts of parvalbumin (PV), a marker for fast-spiking interneurons, in the Git1−/− hippocampal CA1 region, whereas signals for other interneuron markers, somatostatin (SST), calbindin (CB) and calretinin (CR), were normal. n = 3 slices from three mice (WT, KO). *P < 0.05; Student's t test. (e,f) Comparable quantities of tyrosine hydroxylase (TH) signals in brain regions of Git1−/− and WT mice, including caudate putamen (CPu), nucleus accumbens (NAc), substantia nigra (SN) and ventral tegmental area (VTA). n = 3 slices from three mice (WT, KO). Scale bars in a and c, 20 μm; scale bar in e (CPu and NAc), 20 μm; scale bar in e (SN and VTA), 63 μm. Error bars indicate means ± s.e.m.
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References
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    • . . . GIT1 is a multifunctional adaptor protein with several domains, including a GTPase-activating protein domain for ADP ribosylation factor (ARF) small GTPases10; it regulates endocytic traffic of β2-adrenergic receptors and other G protein–coupled receptors (GPCRs)10, 11 . . .
    • . . . One of the candidates was GIT1 (ref. 10). . . .
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    • . . . GIT1 also interacts with diverse signaling and adaptor proteins, including GRK, PIX, FAK, PLC-γ, MEK1, piccolo, liprin-α and paxillin12 . . .
    • . . . Because GIT1, a signaling adaptor, tightly associates with rho guanine nucleotide exchange factors, also known as PIX proteins, in the rodent brain12, 21, we measured quantities of PIX proteins in Git1−/− mouse brains (8–10 weeks) . . .
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    • . . . In rodent brains, GIT1 regulates neurite outgrowth13, 14, spine morphogenesis15, 16, synapse formation15, 16, 17, 18, 19, 20 and synaptic receptor localization of the glutamate receptor AMPA21 . . .
    • . . . Because GIT1 regulates excitatory synapses and dendritic spines in rodents15, 16, 17, 18, 19, 20, 21, we investigated synaptic transmission in the Git1−/− hippocampus . . .
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    • . . . In rodent brains, GIT1 regulates neurite outgrowth13, 14, spine morphogenesis15, 16, synapse formation15 . . .
    • . . . Because GIT1 regulates excitatory synapses and dendritic spines in rodents15, 16, 17, 18, 19, 20, 21, we investigated synaptic transmission in the Git1−/− hippocampus . . .
    • . . . Because the GIT1-PIX-RAC-PAK pathway regulates excitatory synapses and dendritic spines16, 17, we expected a change in excitatory transmission . . .
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    • . . . In rodent brains, GIT1 regulates neurite outgrowth13, 14, spine morphogenesis15, 16, synapse formation15, 16, 17, 18, 19, 20 and synaptic receptor localization of the glutamate receptor AMPA21 . . .
    • . . . Because GIT1 regulates excitatory synapses and dendritic spines in rodents15, 16, 17, 18, 19, 20, 21, we investigated synaptic transmission in the Git1−/− hippocampus . . .
    • . . . Because the GIT1-PIX-RAC-PAK pathway regulates excitatory synapses and dendritic spines16, 17, we expected a change in excitatory transmission . . .
    • . . . However, GIT1 expression is detected at inhibitory synapses17, 21 . . .
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    • . . . In rodent brains, GIT1 regulates neurite outgrowth13, 14, spine morphogenesis15, 16, synapse formation15, 16, 17, 18, 19, 20 and synaptic receptor localization of the glutamate receptor AMPA21 . . .
    • . . . Git1-null (Git1−/−) mice show impaired dendritic outgrowth, reduced spine density, impaired fear responses and reduced adaptation to new and changing environments18, 22 . . .
    • . . . GIT1 deficiency markedly affected survival; ~50% of Git1−/− mice died postnatally, as reported previously18, 22 . . .
    • . . . Because GIT1 regulates excitatory synapses and dendritic spines in rodents15, 16, 17, 18, 19, 20, 21, we investigated synaptic transmission in the Git1−/− hippocampus . . .
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    • . . . In rodent brains, GIT1 regulates neurite outgrowth13, 14, spine morphogenesis15, 16, synapse formation15, 16, 17, 18, 19, 20 and synaptic receptor localization of the glutamate receptor AMPA21 . . .
    • . . . Because GIT1 regulates excitatory synapses and dendritic spines in rodents15, 16, 17, 18, 19, 20, 21, we investigated synaptic transmission in the Git1−/− hippocampus . . .
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    • . . . In rodent brains, GIT1 regulates neurite outgrowth13, 14, spine morphogenesis15, 16, synapse formation15, 16, 17, 18, 19, 20 and synaptic receptor localization of the glutamate receptor AMPA21 . . .
    • . . . Because GIT1, a signaling adaptor, tightly associates with rho guanine nucleotide exchange factors, also known as PIX proteins, in the rodent brain12, 21, we measured quantities of PIX proteins in Git1−/− mouse brains (8–10 weeks) . . .
    • . . . Because GIT1 regulates excitatory synapses and dendritic spines in rodents15, 16, 17, 18, 19, 20, 21, we investigated synaptic transmission in the Git1−/− hippocampus . . .
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    • . . . Git1-null (Git1−/−) mice show impaired dendritic outgrowth, reduced spine density, impaired fear responses and reduced adaptation to new and changing environments18, 22 . . .
    • . . . GIT1 deficiency markedly affected survival; ~50% of Git1−/− mice died postnatally, as reported previously18, 22 . . .
    • . . . The hyperactivity we observed in Git1−/− mice contradicts the recently reported normal locomotor activity of Git1−/− mice22 . . .
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    • . . . GIT1 associates with huntingtin, a protein implicated in Huntington's disease23. . . .
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    • . . . ADHD-affected individuals show cognitive deficits, including behavioral inhibition deficits, deficits in attentional processing and reaction time variability24, 25, 26; similarly, mouse models of ADHD show behaviors consistent with impaired learning and memory27, 28 . . .
    • . . . These findings are analogous to the reported effects of psychostimulants on WT and ADHD model mice27, 28, 29 . . .
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    • . . . ADHD-affected individuals show cognitive deficits, including behavioral inhibition deficits, deficits in attentional processing and reaction time variability24, 25, 26; similarly, mouse models of ADHD show behaviors consistent with impaired learning and memory27, 28 . . .
    • . . . These findings are analogous to the reported effects of psychostimulants on WT and ADHD model mice27, 28, 29 . . .
    • . . . Because mice lacking dopamine transporter show ADHD-like hyperactivity28, thereby pointing to the possibility of abnormal dopaminergic input in the Git1−/− brain, we measured quantities of tyrosine hydroxylase, the enzyme responsible for dopamine production in dopaminergic nerve terminals . . .
    • . . . Our study also suggests that the Git1−/− mouse is a previously unknown mouse model for ADHD, thereby adding to the two known ADHD mouse models28, 29 . . .
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    • . . . These findings are analogous to the reported effects of psychostimulants on WT and ADHD model mice27, 28, 29 . . .
    • . . . Our study also suggests that the Git1−/− mouse is a previously unknown mouse model for ADHD, thereby adding to the two known ADHD mouse models28, 29 . . .
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    • . . . Individuals with ADHD show enhanced electroencephalogram (EEG) rhythms in the theta range (4−8 Hz)30 . . .
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    • . . . As humans and mice show comparable EEG rhythms under normal and pathological conditions31, 32, we measured EEG rhythms in Git1−/− mice . . .
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    • . . . As humans and mice show comparable EEG rhythms under normal and pathological conditions31, 32, we measured EEG rhythms in Git1−/− mice . . .
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    • . . . This is comparable to the psychostimulant-induced normalization of theta rhythms in human subjects with ADHD33. . . .
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    • . . . GIT1 has ARF GTPase–activating protein activity, and its activity toward ARF6, in particular, is crucial for the endocytic regulation of GPCRs10, 35, 36 . . .
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    • . . . Because dopamine receptors are GPCRs and are strongly associated with ADHD37, 38, we tested whether Git1−/− brains show altered ARF6 activity . . .
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    • . . . Because dopamine receptors are GPCRs and are strongly associated with ADHD37, 38, we tested whether Git1−/− brains show altered ARF6 activity . . .
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    • . . . p21-activated kinases (PAKs) act downstream of RAC1 and are implicated in excitatory synaptic structure and function as well as in human brain diseases39, 40 . . .
    • . . . Because Rac1 activates both PAK1 and PAK3 in vitro39, the reasons for this selectivity remain to be determined. . . .
  40. Boda, B.; Nikonenko, I.; Alberi, S.; Muller, D. Central nervous system functions of PAK protein family: from spine morphogenesis to mental retardation Mol. Neurobiol. 34, 67-80 (2006) .
    • . . . p21-activated kinases (PAKs) act downstream of RAC1 and are implicated in excitatory synaptic structure and function as well as in human brain diseases39, 40 . . .
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    • . . . Rac1 plays a crucial part in inhibitory synaptic transmission41, and rodent PAK3 has a role in neurite outgrowth and interneuron migration42 . . .
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    • . . . Rac1 plays a crucial part in inhibitory synaptic transmission41, and rodent PAK3 has a role in neurite outgrowth and interneuron migration42 . . .
    • . . . In addition, PAK3 regulates GABAergic interneuron migration and neurite outgrowth42 . . .
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    • . . . Moreover, alterations in the E-I balance has been postulated to underlie the pathogenesis of neuropsychiatric disorders, including autism47, schizophrenia48, Down's syndrome49, 50, neurofibromatosis type I51, 52, 53 and Tourette's syndrome54 . . .
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    • . . . Moreover, alterations in the E-I balance has been postulated to underlie the pathogenesis of neuropsychiatric disorders, including autism47, schizophrenia48, Down's syndrome49, 50, neurofibromatosis type I51, 52, 53 and Tourette's syndrome54 . . .
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