Download imfar 2007 - Autism Speaks

IMFAR 2007
The scientific study of the brain and nervous system to understand the nature of a disease and its causes,
processes and development is generally termed NEUROPATHOLOGY. This is a report of the oral
presentations and posters relating to the newest neuropathology findings based on investigations of brain
tissue donated after death and how these studies shed light on autism spectrum disorders.
The first two presenters reported on initial findings from the Brain Atlas Project comparing cell numbers and
size in whole brain hemispheres of donors with autism and age-matched unaffected (control) individuals. The
first talk, by Manuel Casanova of the University of Louisville, showed findings of Abnormalities Of Cortical
Minicolumnar Organization In The Prefrontal Lobes Of Autistic Patients; followed by Abnormal Striatal
Circuits In Autism - Links Between Structure And Function by Jerzy Wegiel, New York State Institute for
Basic Research in Developmental Disabilities and Co-PI of the Brain Atlas Project with Christoph Schmitz in
the Netherlands. Figure 1 A-C depicts the brain regions that can be viewed in a single section; there are
approximately 900 sections generated for each brain hemisphere.
Figure 1A shows a brain hemispheric section stained to show neurons. The ‘ribbon’ of blue covering the
entire outer edge of the brain hemisphere is comprised of stained cell bodies in the ‘cortex’. Looking at a
section of the ribbon with higher magnification (Figure 1B), we can see a linear organization from the inner to
the outer edge of the brain. These linear structures are ‘minicolumns’ and as Dr. Casanova explained, they are
evident throughout the brain’s cortex. Figure 1B is important because it represents the replication of his
earlier finding of increased numbers of narrower (smaller width) minicolumns in autism brains. Since these
minicolumns are considered to be functional units in the brain, there are two major questions – what accounts
for the decrease in width and are all regions of the cortex affected the same? The answer based on research to
date is that the number of cells per minicolumn is equivalent; however, the cell size is reduced. And the effect
is more pronounced in the area involved controlling internally generated thoughts or memories (frontopolar
cortex) and that involved in the analysis of socially salient information, including the processing of familiar
faces (anterior cingulate gyrus).
Figure 1A.
Figure 1B.
Control
Autism
Figure 1C. Caudate Nucleus
Dr. Wegiel reported on ‘subcortical’ brain regions involved in social and reward/addiction behaviors (nucleus
accumbens) and rituals/repetitive behaviors (caudate/putamen). The caudate region can be seen in the brain
section (outlined in Figure 1C). Analysis of cells in the target regions showed that the brains of autistic
subjects, relative to controls, have reduced volumes. The cell density of the n. accumbens was reduced 13%
and the numerical density of large neurons was down by 22% in the caudate and by 29% in the putamen. The
reduced volume of the n. accumbens as well as the smaller number of neurons in social brain circuits may be
the substrate of deficits in social interactions. Similarly, the selective deficit in the number of large neurons in
the caudate/putamen could be the component of pathology of striatal dopaminergic circuits linked to repetitive
behavior.
Dr. Izabela Kuchna, also from the NY Institute for Basic Research in Developmental Disabilities, displayed
research results on brainstem that connects the spinal cord to the brain on a poster with the title: Nucleus of Facial
Nerve and Nucleus Olivaris Inferior in Autistic and Control Brain. The impetus for this study was a single
case report published in 1996 of a 21-year-old female with autism with a nearly complete absence of the facial
nucleus (controlling the muscles for facial expression) and superior olive (first major point where auditory
from both ears is combined) input is the first major point where binaural input is combined (Rodier et al., J
Comp Neurol 1996, 370, 247-261). Brain Atlas Project 0.2-mm-thick serial sections were examined and 3-D
reconstruction applied to evaluate the volume of examined structures; a 2-D
representation is shown in Figure 2. The number of neurons was estimated with
unbiased dissector method. Specifically, the nucleus of facial nerve was
examined in 10 brains of subjects who had autism (4-32 years of age) and 9
brains of control subjects (4-59 y) and the volume and the total number of
neurons the autism donors did not differ significantly from those of the controls
(6.8 vs 6.3 cubic mm, and 31.5 vs 27.4 thousand, respectively). Similarly, the n.
olivaris was examined in the brains of 7 subjects who had autism (4-67 years of
age) and 5 controls. The volume and the number of neurons of the nucleus
olivaris inferior principalis were similar in the autistic and control brains (69.8 vs
74.0 cubic mm, and 1.4 and 1.5 million, respectively). These two nuclei appear to
Figure 2
be intact making it unlikely that autism is associated with gross developmental abnormalities
in these brainstem nuclei.
The Brain Atlas Project also provided tissue sections for the investigation of another cortical brain region by
Imke van Kooten, from Maastricht University, the Netherlands. Her poster titled ‘Fewer and Smaller
Neurons in the Fusiform Gyrus in Autism’ focused on the neurobiological basis of face processing. She
investigated the fusiform gyrus as well as the primary visual cortex and the entire cortical gray matter of 7
postmortem brains from autistic patients and 10 controls for volume, neuron density, total neuron number and
mean perikaryal size with high-precision design-based stereology. Compared to controls, brains from autistic
donors showed statistically significant reductions in neuron densities in layer III (-13.1%), total neuron
numbers in layers III and V (-23.7% and -8.3%, respectively), and mean neuronal volumes of layer V (25.9%) in the fusiform gyrus. None of these alterations were found in the primary visual cortex or in the
cerebral cortex making the face processing area of the brain a target of ongoing study of the cellular basis of
abnormalities in face perception in autism.
BRAIN TISSUE GENETICS
The numbers of cells in a particular brain region and their size at any given time and their synaptic
connections with other cells is determined by genes and any environmental impact during pre- and post-natal
development. Several researchers reported on the assessment of gene activity in various parts of the brain as a
start in defining changes in autism brain tissue compared to that from unaffected donors. Three oral and two
poster sessions devoted to autism brain tissue and molecular genetic findings.
SLC25A12 Expression Is Up-regulated In Autism Prefrontal Cortex and Associated With Neurite Outgrowth.
Nicholas Ramoz, INSERM U675, France. Dr. Ramoz provided background for his group’s study: multiple
genomic screens have shown evidence suggesting linkage to chromosome 2q31-q33, which includes the
SLC25A12 gene. An association between autism risk and two single nucleotide polymorphisms (SNPs) in
SLC25A12 was reported in an independent data set of 327 families with autistic offspring. The association of
the gene SLC25A12 with autism is controversial so the strategy was to directly evaluate and compare two
brain areas for expression of the gene and analyze the results again a background of the expression of that
gene in human development and a mouse model. They found that the SLC25A12 level of transcript was
significantly higher in the post-mortem BA46 frontal cortex region of autistic subjects than in controls. In
contrast, no difference of SLC25A12 expression was observed in the granule cells of cerebellum lobule VI
between patients and controls. During human prenatal development, neurons over-expressing the gene
product had significantly longer dendrites and more branches than neurons transfected with the control GFP
vector. The conclusion is that SLC25A12 over-expression may be involved in autism pathophysiology by
changing neuronal morphology in specific brain subregions. More work in this area is necessary to see if or
how this gene contributes to autism.
15q11-13 GABAA Receptor Genes Are Normally Biallelically Expressed in Brain yet Are Subject to
Epigenetic Dysregulation in Autism is the title of a talk by graduate student Amber Hogart of the University
of California, Davis. An outstanding representative of the student-researcher, Amber explained that maternal
duplication of a segment of Chromosome 15 called 15q11-13 occurs in 1-2% of cases of autism. 15q11-13 is a
complex locus containing ‘imprinted’ genes for other disorders such as Prader-Willi and Angelman syndrome
and has a cluster of three genes that make receptor subunits for the GABAA receptor controlling inhibitory
transmission in the nervous system. It is not known if these subunit (GABR) genes, GABRB3, GABRA5, and
GABRG3 are imprinted too. ‘Imprinting’ is a form of control of gene expression and operates in Rett
disorder; therefore, Amber and her group designed an experiment to determine conclusively if the 15q11-13
GABR genes are normally imprinted in human brain, and examine expression of these genes in autism, Rett
syndrome and non-affected ‘control’ brain samples. Each gene has two alleles – one on the chromosome
from the father and one from the chromosome from the mother. Equal biallelic expression of GABR genes
was observed in 21 heterozygous control brain samples, demonstrating that these genes are not normally
‘imprinted’, a condition that would result in either reduced or over-active production of the gene’s mRNA.
Quantitative RT-PCR analysis of brain samples with paternal and maternal 15q11-13 deletions revealed a
paternal expression bias of GABRB3 compared to the equal allelic expression in control samples.
Interestingly, 4 of 8 autism, and 1 of 5 Rett syndrome brain samples showed loss of biallelic expression of one
or more GABR gene, suggesting that epigenetic dysregulation of these genes is common in autism-spectrum
disorders.
Jane Yip, a post-doctoral student at Boston University, MA, gave a talk on the Studies of GAD65 mRNA
Levels in the Deep Cerebellar Dentate Nuclei in Autism. She described work on the dentate cerebellar nuclei
(CN), brain structures that provide information linking the cerebellar cortex to higher cognitive centers,
previously reported to be dysfunctional in autism. The study was undertaken to see if inhibitory circuits based
on GABAergic neurotransmission in the CN are involved in neuropathology observed in the CN in autism and
focused on the genetic message (mRNA) the makes GAD65, one of the isoforms of the GAD enzyme in the
CN converting glutamate to GABA. The level and distribution of the GAD65 mRNA in major dentate CN
cell groups were analyzed. These cell groups are characterized by small or large cells shown in Figure 3 and
she found a significant reduction in GAD65 mRNA levels in the dentate CN of the medium-large cell
population in the autistic group compared to the control group (p=0.03; independent t-test); there was no
significant difference in the small cell population of the dentate CN between the two groups. The decrease in
GAD65 mRNA levels in the mediumlarge cells of the dentate CN in the
Differential sizes of neurons in the dentate nuclei
autism brain samples suggests that a
subset of medium-large dentate CN
neurons contain GABA and colocalize GABA with another
transmitter such as glutamate. These
findings may have effects on a specific
portion of the CN circuit that provides
inhibitory feedback to Purkinje cells,
inferior olivary neurons, and perhaps
the thalamocortical tract.
Left panel: GAD65 mRNA in a medium-large dentate neuron in an adult control case.
Right panel: Similar labeling in the cell body of a small dentate neuron.
Dr. Yip’s mentor and laboratory head,
Dr. Gene Blatt, proposed an
Figure 3
explanation for involvement of the
brainstem-cerebellar circuitry in
autistic behaviors based on collective
findings from past work on this problem. In An Emerging GABA/Glutamate Hypothesis of Cerebellar
Dysfunction in Autism, he reviewed evidence that a disruption of innervation from the brain stem to the
cerebellar cortex may lead to compensatory changes in two key neurotransmitter systems in the cerebellar
cortex, GABA and glutamate. Drs. Blatt and Yip are developing a circuitry model that suggests alterations in
climbing fiber input to Purkinje cells, possible altered function in the remaining Purkinje cells in the
posterolateral cerebellar hemisphere resulting in abnormalities in at least one subset of dentate neurons. Thus,
alterations in the output of the cerebellum to higher structures may contribute to motor and/or cognitive
behavior deficits in autism.
Another experiment from the Blatt lab was depicted in a poster Altered GABA-A Receptor Binding in the
Anterior Cingulate Cortex in Autism, discussed by doctoral graduate student Adrian Oblak. The anterior
cingulate cortex (ACC) is a current target of investigation in several laboratories due to observed changes in
autism brains and its role in mediating executive function, affect, and socio-emotional behavior. The
distribution of GABA-A receptors and benzodiazepine binding sites in the anterior cingulate cortex of adults
with and without autism was investigated. Benzodiazepine molecules (think valium) bind to specific
receptors on cells and activate another receptor, GABA-A, that receives the inhibitory neurotransmitter
GABA. The pharmacological benefit of the benzodiazepine family of sedative drugs is thought to derive from
the potentiation of GABA transmission. She found a significant reduction in the number of GABA-A
receptors in the superficial and deep layers of the ACC and a reduction in the density of benzodiazepine
binding sites in the two layers (with no change in binding affinity - meaning that the receptors, although
reduced in number, were able to bind the molecules normally). The experiments add to the building evidence
that the inhibitory GABA system is compromised in the ACC in autism possibly contributing to the socialaffective behavioral alterations observed in this disorder.
Brain Donation. We wish to thank each and every family who supported the brain donation of a
loved one for the purpose of understanding autism spectrum disorders, including those without known
disorders. Brain tissue donated for research requires speedy yet careful recovery of tissue within 24
hours of death. The ATP collaborates with the Harvard Brain Tissue Resource Center, the main
repository for autism brain tissue and provides a clinical coordinator to provide Autism Tissue
Program registrants with caring, sensitive and expert help in the brain tissue donation process. In the
event of a potential donation, relatives, friends, educators, researchers or clinicians are advised to call
the same 24 hour hotline at 1-877-333-0999. They will then be put in touch directly with a brain bank
representative who will then contact a local pathologist to assist with tissue recovery at the area
hospital. Even if a person is not pre-registered, donation can be arranged with this phone call. ATP
assumes all costs of recovery and brain donation does not interfere with funeral or memorial
arrangements. Complete information and secure online registration for future donation is available on
the ATP website, http://www.autismstissueprogram.org.. The hotline at 1-877-333-0999 may also be
contacted for further information or materials regarding the program.