Elevated urinary P-Cresol in small autistic children: Origin and consequences
Antonio M. Persico, M.D.
Laboratory of Molecular Psychiatry & Neurogenetics, University Campus Bio-Medico, Rome, Italy
This research project aims at continuing and completing the following three studies:
(1) assessing gut permeability, urinary p-cresol, fecal c. difficile/toxin A/calprotectin and urinary cotinine in approximately 50-60 individuals should suffice, if our results continue to show no correlation between these three parameters. We shall also try to implement a fecal metabolomic approach to assess gut microbiota beyond c.difficile, in order to exclude infection by other cresol-producing bacteria and to point convincingly toward environmental exposure as the cause of elevated urinary p-cresol in autism;
(2) conduct a replication study by recruiting at least other 13 case-control pairs, reaching a final N of at least 30 autistic individuals and 30 matched controls, equally split between age <8 and age > 8;
(3) performing a complete battery of tests assessing motor (Open Field Test, Rotarod Test and Grooming activity), cognitive (Object Recognition Test, Spatial Novelty Test) and emotional (Porsolt’s Test, Plus Maze, Social Novelty Test) responses to acute administration of p-cresol in BTBR mice, which are regarded as one of the most reliable rodent model of autism. Behaviors will be automatically analyzed with EthoVision XT System (Noldus, Wageningen, Netherlands). Neurochemical analyses will be performed on the brains of these mice, to assess brain region-specific imbalances in major neurotransmitters.
Metabolic factors affecting Gamma synchrony
Manuel Casanova, M.D.
University of Louisville
Richard Deth, Ph.D.
Many autistic individuals have difficulties in putting together the different parts of a sensory experience. As an example, they remember faces by component features (e.g., lips, type of glasses worn) rather than by acquiring familiarity with the whole, an ability that is based on the integrity of fast-spiking interneurons. Many studies presently suggest an excitatory-inhibitory imbalance in the cortex of autistic individuals based on a defective peripheral surround to the cell minicolumn, the location where most inhibitory neurons are located. We have found that both Transcranial Magnetic Stimulation (TMS) and neurofeedback improve the ability of autistic individuals to “bind” together aspects of sensory perception. In this study we will use both rTMS and neurofeedback synergistically in a clinical trial, and use an oxidative panel to screen for a possible biomarker capable of predicting outcome. It is hypothesized that fast-spiking interneurons have a higher metabolic rate than other neurons (e.g., pyramidal cells) and their malfunction could provide for abnormal binding of sensory experiences as well as an abnormal metabolic panel. If positive, our results will be pursued by studying specific fast-spiking interneurons (e.g., Neuregulin-1 expressing cells), which provide a well-known risk factor of autism. This study provides a unique opportunity to evaluate the relationship between neurological function and antioxidant status.
To study urokinase-type plasminogen activator plasma concentration and its relationship to Hepatocyte Growth Factor (HGF) and GABA levels in autistic children
A.J. Russo, Ph.D.
Hartwick College, Oneonta, NY
Excitatory neural activity in the mature cerebral cortex is modulated by local GABAergic interneurons. These neurons originate in the ventral telencephalon and migrate to populate the dorsal forebrain. Disruption of the GABAergic interneuron population during development results in improper circuit formation and seizures in humans and mice. Hepatocyte growth factor/scatter factor (HGF/SF) is expressed in the prenatal forebrain and regulates neuronal migration. Latent HGF/SF is activated by serine proteases, including urokinase-type plasminogen activator, uPA. When uPA is bound to its receptor, uPAR (also known as Plaur, as the gene is Plaur), the protease activity is strongly accelerated. Loss of Plaur leads to the reduction of HGF/SF in the embryonic forebrain, interneuron deficits, and subsequent spontaneous seizures. Functional polymorphisms in the plasminogen activator, urokinase receptor (PLAUR) gene, the human homolog of the mouse uPAR gene, like those found in MET, increase the risk for ASD. Also, Endogenous postnatal supplementation of HGF/SF ameliorates the interneuron defects in B6.129 – Plaurtm1/Mlg mice and alters electrophysiological activity to approach normalcy, suggesting that HGF supplementation may affect behavior in autistic individuals.
We found decreased levels of HGF in autistic children and adults with depression, anxiety, bipolar disorder and schizophrenia and suggest that this is associated with altered levels of urokinase type plasminogen activator (uPA).
We intend to measure uPA, HGF and GABA levels in autistic children to see if uPA levels correlate with HGF and GABA levels. We hypothesize that decreased levels of HGF will be associated with altered uPA, and increased GABA, suggesting a role for uPA in the etiology of autism.
To study the relationship between low GAD2 levels and anti-GAD antibodies in autistic children
A.J. Russo, Ph.D.
Hartwick College, Oneonta, NY
Glutamic acid decarboxylase (GAD) is the rate-limiting enzyme in the synthesis of the widespread inhibitory transmitter γ-amino butyric acid (GABA) by catalyzing the decarboxylation of glutamate. Antibodies against glutamic acid decarboxylase 65 (GAD65 or GAD2) have been detected in the serum of patients with any of several neurological disorders, including depression, bipolar disorder and schizophrenia. They have also been found recently in individuals with autism and ADHD. The understanding of the effect of these antibodies against GAD65 has not yet been clarified.
There is much support for the role of GABA in the etiology of autism. Recent research has shown that hepatocyte growth factor (HGF) modulates GABAergic inhibition and seizure susceptibility (6-9). Our lab has been studying the relationship between HGF and GABA levels in autistic children.
We recently determined plasma levels of HGF, GABA, GAD2 as well as symptom severity in autistic children and neurotypical controls (supported by ARI grant; To study the Relationship Between Decreased Hepatocyte Growth Factor (HGF) and Glutamate Excitotoxicity in Autistic Children).
We previously reported that autistic children had significantly decreased levels of HGF. In preliminary research, the same group of autistic children had significantly increased plasma levels of plasma GABA (p=0.002) and decreased HGF levels correlated with these increased GABA levels (r=0.3; p=0.05), supporting a relationship between HGF and GABA. In this same study, GABA levels correlated with increasing hyperactivity (r=0.4; p=0.01) and impulsivity (r=0.3; p=0.04) severity.
In preliminary data, we also found GAD2 plasma levels were significantly lower in individuals with autism (N=27) compared to neurotypical controls (N=12) (p=0.0001), and that plasma levels of the inflammatory marker HMGB1 correlate significantly with the GAD2 levels in these same individuals (r=.6; p=0.003), supporting the premise that the etiology of autism is associated with inflammation and might be associated with glutamate excitotoxicity and GABA mis-modulation, due in part to GAD2 deficiency. It is possible that autoimmune response to GAD2 (presence of ant-GAD antibodies) is responsible for binding and lowering available GAD2 in some autistic children. This study is designed to begin to determine if plasma levels of antibodies to GAD2 are associated with the observed low GAD2, GABA, and/or HGF plasma levels, as well as symptom severity in autistic children.
Brain mitochondrial abnormalities in autism
Abha Chauhan, Ph.D.
NYS Institute for Basic Research in Developmental Disabilities
Emerging evidence suggests role of mitochondrial dysfunction and oxidative stress in the development of autism. Mitochondria play crucial roles in energy production, free radical generation and apoptosis. Mitochondrial energy production is coupled with electron transport chain (ETC) and tricarboxylic acid (TCA) cycle. Although several case reports have implicated mitochondrial dysfunction in autism, there has been little systematic evaluation of mitochondrial abnormalities in autism, particularly in the brain. We hypothesize that abnormal mitochondrial dynamics (fusion and fission) and biogenesis, and increased apoptosis via mitochondrial pathway may be involved in the etiology of autism. We have recently reported brain region-specific deficit in mitochondrial ETC complexes and oxidative stress in selective brain regions i.e., the cerebellum, frontal cortex and temporal cortex in autism. The goal of this project is to identify mitochondrial abnormalities in autism by studying postmortem brain samples from subjects with autism and their age-matched control subjects. This will be achieved by studying (a) mitochondrial fusion and fission, (b) mitochondrial biogenesis, and (c) apoptosis via mitochondrial pathway (levels of apoptosis-inducing factor and cytochrome c). The results of this study will provide evidence as to whether abnormalities in mitochondrial functions are associated with autism. This study will also suggest whether a subset or a majority of autism subjects has mitochondrial abnormalities. The results of this project may help in the design of biochemical targets for early diagnosis, assessment of the risk for regression or development of autism, and therapeutic interventions for autism.
Autism spectrum disorders –inflammatory subtype: molecular characterization
Harumi Jyonouchi, M.D.
University of Medicine & Dentistry of New Jersey
Clinical presentation of autism spectrum disorders (ASDs) differs markedly in each child and many ASD children also suffer from various other medical conditions, with gastrointestinal (GI) symptoms one of the most common. We have previously shown that in young ASD children, GI symptoms can be attributed to non-IgE mediated food allergy (NFA), since GI symptoms resolve with a restricted diet (RD), i.e., avoidance of offending foods in ASD/NFA children. However, some ASD/NFA children do not respond to the RD. These ‘poor responders’ are characterized by fluctuations in behavioral symptoms and cognitive skills triggered by illnesses such as viral infection, along with poor responses to pharmacological/behavioral interventions.
By studying peripheral blood (PB) cells called monocytes (Mo) obtained from these ASD children, we found altered responses in innate immunity, the immune defense system that regulates initial immune responses. Innate immune responses alert the brain of immune-activating events, affecting the brain’s function. Recently, a genetic study of autistic brains revealed that there is an ‘immune activation module’ which is up-regulated in autism. This finding indicates that inflammatory responses in other organs can affect the brain, in what can be called ‘immune/inflammatory autism’. ASD children with the above-described findings are likely to fall into this subtype, and categorized as the ASD-inflammatory subtype (ASD-IS) in this study.
Despite evidence that suggests a role of the immune system in some ASD subtypes, previous studies addressing immune abnormalities in ASD have been inconclusive. This is in large part due to the failure to separate ASD children with immune/inflammatory components from those without. ASD-IS children have distinct immune abnormalities that can be tested using objective laboratory assays, and the results can be assessed in comparison with changes in their neuropsychiatry symptoms. This makes ASD-IS children ideal for studying the role of the immune system in ASD. This study focuses on identifying biomarkers for ASD-IS children in PB Mo, in comparison with ASD/normal controls. Analysis of immune parameters will be done repeatedly over the course of the study to allow for comparison with changes in behavioral symptoms in ASD-IS children. Our long-term goal is to find markers that permit early detection of ASD-IS children, and to improve the disease outcome.
Using high-definition fiber tracking to define developmental neurobiologic mechanisms & a neural basis for behavioral heterogeneity
Marcel Just, Ph.D.
Carnegie Mellon University
fMRI imaging studies have demonstrated lower functional connectivity (a measure of the synchronization of the activation between pairs of brain areas) in autism in many high-level cognitive and social tasks, suggesting impaired communication between frontal and posterior brain areas. At the same time, diffusion tensor imaging (DTI) of the white matter that provides the communication pathways between brain regions has consistently shown widespread abnormalities in white-matter regions in autism. Our project is attempting to map the axonal wiring diagram of the white matter in autism. Within the last year, we have made transformative technological advances in High Definition Fiber Tracking (HDFT) that permit tracing of individual fiber tracts at previously inaccessible levels of precision and accuracy.
This project uses this fine-grained mapping of cortical connectivity to directly test the hypothesis that dysregulated fiber-level connectivity among brain areas plays a major role in the pathophysiology of autism. We are characterizing white matter tracts and topography in a sample of controls and individuals with autism ages 18 to 65 years, and comparing these neural properties to cognitive and behavioral measures of functional outcome, emotion regulation, problem solving, and social and language comprehension. The project has the potential to provide an integrated account of the behavioral symptoms of autism explained in terms of its anatomical and brain function bases, and to provide increased specificity and sensitivity of autism diagnosis by the use of the imaging biomarkers.
Regressive autism as an infectious disease: Role of the home as an environmental factor
Sidney Finegold, M.D.
UCLA/VA Medical Center, Los Angeles
The intent of the study is to do bacteriologic studies on approximately four subjects with regressive autism and four controls; these studies will include both examination of stools of the children and siblings and parents, as well as their fingers, and then an extensive study of surfaces and fomites in the home environment. Finally, we will compare like isolates from the subjects and the environment to verify that they are identical (or not, as the case may be). This would be a major step in verifying the epidemiology of regressive autism.
The effects of the Hane Face Window© on perceptual processing of children with Autism Spectrum Disorders (ASD)
Albert Yonas, Ph.D. and Sherryse Corrow, M.A.
University of Minnesota
Gaze avoidance, and particularly fixation on the internal features of a face such as eyes, is a diagnostic criterion for Autism Spectrum Disorder (ASD); it is believed to contribute to the development of social deficits. The Visual Perception Lab at the University of Minnesota is exploring one technique to increase attention to faces, the Hane Face Window. This window, developed by Ruth Elaine Hane, occludes all parts of the face with the exception of the eyes, nose and mouth, increasing the likelihood that viewers will fixate on the internal features of faces. Wilson (2010) argues that Hane Face Window may reduce social relevance and the social fear evoked by a face. The laboratory is currently testing the hypothesis that the Hane Face Window will increase the ability of children with ASD to fixate on the internal features of faces, as compared with a control group of typically developing children (ages 7-14). Furthermore this change in attention will improve the ability to recognize a face and perceive the direction of gaze and infer where the individual’s attention is focused. Following gaze is one component of joint attention in which children with ASD are deficient. In the study, performance is assessed in a face recognition task and in a gaze-following task in which a face is presented with the Hane Face Window without the window. In addition, an eye tracker is being used to collect information on whether the Hane Face Window will increase the number and duration of fixations on the eyes and central region of the face.
Electrophysiological and behavioral outcomes of auditory integration training (AIT) in autism
Estate M. Sokhadze, Ph.D., Manuel Casanova, M.D., and Allan Tasman, M.D.
University of Louisville
The proposed study aims to understand the abnormal neural and functional mechanisms underlying sound-processing distortion in autism by incorporating neurophysiologic and behavioral studies, and measurements of auditory attention in several different auditory tests. The study will use Berard’s technique of auditory integration training (AIT) to improve sound integration in children with autism. It is proposed that exposure to 20 thirty-minute AIT sessions (total 10 hours) will result in better performance on auditory attention and perception tasks, and will lower anxiety as indexed by a profile of post-AIT autonomic measures.
We propose to test 30 children with autism in task using auditory stimuli in perception and attentional tests. These behavioral and psychophysiologic studies will be carried out by using electroencephalogram (EEG), and dense array even-related potentials (ERP). During AIT or during auditory tests autonomic measures (HR, HRV, skin conductance, respiration, skin temperature) can also be monitored. The behavioral studies in EEG/ERP test mode will be carried out by using equipment that measures both reaction time and accuracy in high functioning autism participants. The measurement of attention and perception will be carried out using different modifications of auditory tests in low-functioning individuals (capable to tolerate EEG recording), in particular these auditory tests will not require any motor responses.. The results of the proposed study will aid in our understanding of specific neurocognitive deficits associated with developmental abnormalities within cortical circuitry related to hearing and sound processing, test whether performing AIT course may enhance auditory integration process and thereby contribute to understanding the brain substrates of dysfunctions typical for autism, and result in behavioral improvements.