Using Short Hairpin RNA (shRNA) in Order to Combat the Negative Effects of Autism Spectrum Disorder

By: Ty Krug, Trever Barnes, Chris Spates, and Garrett Briggs

 

Autism Spectrum Disorder (ASD) is known as a wide-spectrum developmental disorder that causes problems with communication, social, verbal, and motor skills. It is said to be found in 1 in every 68 children, and no two people will have the same effects from autism, as the symptoms vary from person to person (Wright 2015). At this point in time there are no direct causes for autism though many seem to believe it can be caused by genetic, epigenetic, and environmental factors. It is known however, that people with autism have abnormal brain structure and functionality which usually begins before the age of 3. Although it cannot be determined, researchers have narrowed down a possible cause of autism residing in the cortex of the human brain.  ASD is classified as a neurodevelopmental disorder, defined by impairments in social interaction, deficits in verbal and nonverbal communication, and the display of stereotyped and repetitive behaviors (Aigner et al. 2013).  During normal brain development, a burst of synapse formation occurs in infancy. This is particularly pronounced in the cortex, which is central to thought and processing information from the senses.  As an individual gets older, a process in the brain called pruning occurs, where many of the brain synapses are trimmed, leaving only the major synaptic pathways.  Through research, it can be determined that patients with ASD did not go through the pruning process that normal infants did (Wright 2015).

There are many human neurodevelopmental toxicants that have health impacts that are supported through numerous research. These toxins/metals include Mercury, Lead, Arsenic, Fluoride, and Manganese (Kalkbrenner et al. 2014). All of these toxicants can lead to negative effects on the developing nervous system which can result in a loss of IQ points and behavioral problems. Research shows that all of these toxicants can have a negative impact on the human neurological system even at typical exposure levels (Talbott et al. 2015). This can be dangerous for mothers who are pregnant with a baby in the critical developmental stages, and could possibly have a direct affect on the outcome of autism. What is challenging about these toxins is the way people can become exposed to them. Exposure can be from ingestion or even inhalation and overtime can cause effects that last a lifetime. Another form of toxin that has been shown to directly affect autism in the prenatal stages is pesticides(Shelton et al. 2012). Pesticides can lead to neurotoxicity, and unfortunately have been detected in over 97% of biological samples (Kalkbrenner et al. 2014). Pesticides such as organophosphates and pyrethroids have the ability to pass through the placenta and then continue on through the blood-brain barrier which can cause neurodevelopmental problems in a fetus or infant. Infants exposed to these pesticides are 60% more likely to end up with a neurodevelopmental disorder such as ASD. Though these toxins and pesticides discussed above are vastly different they ultimately are shown to have an effect on the prenatal fetus leading to autism (Shelton et al. 2012). It is hard to say specifically how much these factors directly impact the outcome of autism due to the challenge in measuring exposure concentrations and sample sizes, however, it is proven that prenatal exposure to the chemicals thalidomide, folic acid, and valproic acid due lead to an increased risk of autism (Tijdschr 2014).

The cellular phenotype for ASD contains I characteristics which are not severe enough to present definite or readily observable conditions. The combination of this broad variation of phenotypes and a high rate of agreement in identical twins suggests a large number of genetic and environmental biasing factors (Belmonte 2004). According to some estimates, mutations in as many as 1,000 genes could play a role in the development of these disorders (Farley 2013).  

The phenotypes of autism are the primary indicator of the disorder due to the fact that there is not enough known about the genetic factors to diagnose Autism based solely off of DNA.  The “spectrum” of ASD refers to the phenotypic spectrum characterized by behavior studies.  A compilation of data conducted by Biomedical ontologies recognize 283 concepts of autism coming off of the the main three branches (Mccray et al. 2014). The figure to the left illustrates some of the initial branchings that continue to branch until they reach very specific concepts.  Example concepts include “Emotional regulation and control- directing and governing one’s own emotions” and “Self injurious behavior-  behaviors in which a person intentionally hurt or harm themselves.”  (Mccray et al. 2014)  There is a standard test to measure the social and communication deficits in ASD, called the Autism Diagnostic Observation Schedule-Generic (ADOS-G) (Lord et al. 2000).

Many genes can contribute to the phenotypes shown above; for example, in one study, scientists identified 107 genes that show a strong correlation with autism.  Most of the genes encode proteins for synaptic formation, transcriptional regulation and chromatin-remodelling pathways.  It is a loss of function in these genes that seems to be related to a person having ASD (De Rubeis et al. 2014).  The chromatin remodelling pathways deal with regulation of gene expression (Allison and Milner 2004).  A malfunction in one of these genes could have an affect on many genes because the protein from this gene is used to regulate the expression of many genes.  According to Dr. Brose,  the synaptic-forming proteins are called neuroligins, and they are responsible for the maturation of the synapses in the brain.   Without neuroligins, the synapses do not have enough receptor proteins, and therefore  have a reduced ability to pass signals (Brose 2006).  The proteins involved with autism are the transcriptional regulation proteins.  A loss of function in these proteins would result in wide-scale mutations in DNA that would not necessarily be related to one gene.

Many genes are linked to ASD, and scientists have yet to discover them all and create a comprehensive list.  It is for this reason that they are often classified into general types by the proteins that they code for, as they are in the previous paragraph.  That being said, this section will explore a few specific genes that have shown to be related to ASD, and their direct effects on the body.  One study shows the relationship between the oxytocin receptor gene (OXTR) and specific phenotypes of ASD: social impairment and repetitive behavior (Yu et al. 2015).  Two single nucleotide polymorphisms (SNP) in the 3’UTR region of the OXTR gene are what causes these two common phenotypes of ASD (Harrison et al. 2015).   Another example of a specific gene  that is strongly related to ASD is the gamma aminobutyric acid receptor (GABA) subunit genes (Ma et al. 2014).  This gene creates the receptor subunit, which controls synaptic inhibition in adults.  A mutated GABA gene does not allow for inhibition of excitement-inducing chemical signals, which contributes to the phenotype of inattention and restlessness in autistic people (Bowery and Smart 2006).

Epigenetics have also been proven to play a large role in autism. However, like other factors, epigenetics can not be summed up in one distinct cause and effect. It can affect the genetic expression in several different ways. Two prevalent ways that epigenetic changes in individuals with autism differ with normal people are through autosomal monoallelic expression and X-inactivation (Ben-David et al. 2014). In an experiment conducted in 2014 by several doctors at the Hebrew University of Jerusalem they explored the role that epigenetic factors played in autism (Ben-David et al. 2014). What they discovered was that individuals with autism rarely express the same genetic mutations (Wong et al. 2013). They also were able to determine that in most cases the regulation of epigenetic factors which restrict genetic expression were not present, in one case allowing up to 10 times more monoallelic expression than in the average human being (Ben-David et al. 2014). This often resulted in a larger brain mass and several different levels of mental retardation ranging from mild to severe (Wong et al. 2013). X-inactivation is also affected in individuals with autism. It has been demonstrated that several epigenetic factors do play some effect on the outcome of an individual’s autistic traits. However with current technology there is no real way to test all the different epigenetic factors that play a role in autism are and how significant they are at this current time (Ben-David et al. 2014).  Because Autism Spectrum Disorder (ASD) has such a vast range of causative factors; with epigenetic, genetic, and environmental influences, it can most clearly be explained by starting with the more clearly defined phenotype, and ending with the almost limitless possible causative factors.  

According to one study, the autism spectrum encompasses 283 distinct phenotypic components, and a person suffering from the disorder will usually display several of them (McCray et al. 2014).  They can be generalized into these categories: impairments in reciprocal social interaction, communication deficits, and restricted patterns of behavior; all of which are neurobiological symptoms that can be related to synaptic issues  (Yu et al. 2015).  

There are many genes which could contribute to the cause of ASD collectively, as they are involved with neurological synapses and leukocyte production.  Genes such as NLGN3 and NLGN4 contribute to the formation of the central nervous system, as well as the surface protein to neural cells (Jamain et. al.).  In regards to leukocyte production with Autism, genes such as HLADR4 and HLADR14, both located on chromosome 6, regulate for a non specific and uncharacterized brain protein which is responsible for the production of autoantibodies (Torres et al. 2012).  Although these specific genes show evidence to being causative to ASD, researchers have narrowed down the underlying cause residing in the synaptic pathways of the brain (Auerbach et al. 2011).

When attempting to narrow down which genes are causative to ASD, we identified one gene in particular, Cx3CR1, which had a significant effect on the brain and their synaptic pathways.  Autism Spectrum Disorder is caused by an excess of brain synapses, most of which are weak and immature (Farley 2013).  In a normal functioning human brain, a child going through infancy expresses this Cx3CR1 gene which is located on chromosome 3, and produces a protein called fractalkine.  Fractalkine, in infants, prunes out the weak and immature brain synapses, leaving strong single firing pathways for brain synapses.  In a child with Autism Spectrum Disorder, a developing child goes through a mutation called a microdeletion, which is an unexpected partial cutting of a chromosome.  In this microdeletion, the Cx3CR1 gene is cut unexpectedly from the child, leading to a microglia deficiency and no production of the fractalkine protein (Bifone et. al 2014).  In absence of the fractalkine receptor protein,  weak and immature synapses are not pruned throughout the autophagic pathways as microglia are not signalled to prune, leading to excess firing of weak synapses and inhibited growth of the more developed and stronger ones (Zhan et al. 2014).  

In a study performed on mice, results showed that autistic-like synaptic deficiencies were directly correlated to levels of mTOR in synaptic pathways, resulting in impaired autophagy.  These results displayed excess mTOR inhibiting synaptic pruning within mice in their first week of development ( Tang et. al. 2014).  Although research was inconclusive in connecting mTOR with the fractalkine protein, their correlation cannot be ignored.  Because excess mTOR results in the blocking of synaptic pruning, a potential treatment would be to inhibit mTOR.  Studies have been done previously to inhibit mTOR production, and mice were injected with the mTOR inhibitor rapamycin.  Following the injection, mice had increased brain function as learning and memory impairments were reversed (Ehninger and Alcino 2011).  This study draws results directed towards the treatment of Autism, as it shows brain deficiencies due to stimulation of weak and immature pathways that were not pruned can be reversed and no irreversible structural damage was caused due to the defect.

In order to correct the symptoms caused by autism it is necessary to combat the loss of fractalkine production which results from the mutation of the Cx3CR1 gene (Bifone et. al, 2014). The loss of the fractalkine protein would result in an excess of synapses because the normal pruning would not occur; meaning that unnecessary synapses would remain, and there would be overstimulation of neuronal signalling. We propose that the solution to the problem is to implant a SRPX2 gene inhibitor as they did in the study conducted by (Sia et al 2013). The inhibitor is a short hair pinned RNA (shRNA).  The SRPX2 gene is responsible for the formation of new synapses.  In their study, they showed that the “SRPX2 blocking compound”, when injected into the mice, resulted in less synaptogenesis.  Humans have the same gene (Sia et al. 2013).  Our treatment would involve injecting the SRPX2 blocking compound into someone with ASD postnatally.  Treatment would occur postnatal because basic synaptic formation would have to take place. However, when symptoms of autism begin to appear, it would be beneficial to reduce the number of synapses being formed to lower it to a normal level. Immediately following birth treatment would begin by way of inserting the blocking compound into the baby in order to regulate the proper amount of synapse activity needed for normal brain function.  Doing so would solve the underlying problem of ASD; too much synaptic activity.  

In order to reduce the phenotypic symptoms of ASD the child would be infected with a virus containing shRNA which would directly limit the amount of synapses. However, the gene could not be fully repressed therefore the shRNA levels inserted into the child would have to be regulated. If the levels are too low then no change will occur yet if the levels are too high it could become toxic within the child. Fortunately, in their study it was proven that you can regulate the proper amount of shRNA depending on what level of expression would result in a more healthy phenotype.

Overall, it is important to point out that Autism is still being researched heavily today. It is unclear what causes each individual mutation that leads to autism, however, genetic research points toward the suppression of brain synapses will minimize the phenotypic problems seen in autism patients. Researchers have displayed positive results from inserting a drug, rapamycin, into the brain of mice which enables a gene to suppress excess mTOR, as it has improved brain function and memory retention, however they have still yet to find a connection to mTOR and excess synapses in autism.  Because there are hundreds of different genes which are causes for ASD, there is no defined pathway which the genetic disorder follows on a case by case basis.  Because of this, not all cases of Autism are directly affected by excess brain synapses.We propose that, in order to show the symptoms of ASD, one must be influenced in each of the following areas: epigenetic, genetic, and environmental.  More specifically we believe that pesticides and pharmaceuticals play the main role in linking the epigenetic, genetic, and environmental factors together that directly relate to prenatal effects causing autism.  However, the proposed treatment plan can improve upon a patient’s symptoms, and could potentially allow them to enjoy a more normal brain function.

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