A hidden disability? - Automatic identification of autistic children based on appearance reaches 94% accuracy. Spectrum Needs assessed in a small trial. Bullying in ASD. TCF20 and GABAa receptors. Special educational needs – not so special any more.

 

Today’s post is a summary of a small part of the recent autism research. I am constantly amazed how much autism related research is churned out every day. To anyone who says more autism research is needed, just take a look at how much there already is !!  

 

Facial recognition of Autism?

Those working every day with special needs children have long known that you can pretty quickly spot a child with autism, without any lengthy diagnostic procedure.

Some advocates like to see autism as a hidden disability and believe you cannot “look autistic.” They had better not read this post.

I did write about facial recognition of single gene autisms and rare diseases where a commercialized product (Face2Gene) can now identify 200 conditions with 91% accuracy. This is from a single photo of the face. 

Now Chinese researchers have produce software that can predict autism in pre-schoolers with 94% accuracy based on automated analysis of a video.

Risk assessment and automatic identification of autistic children based on appearance

The diagnosis of Autism Spectrum Disorder (ASD) is mainly based on some diagnostic scales and evaluations by professional doctors, which may have limitations such as subjectivity, time, and cost. This research introduces a novel assessment and auto-identification approach for autistic children based on the appearance of children, which is a relatively objective, fast, and cost-effective approach. Initially, a custom social interaction scenario was developed, followed by a facial data set (ACFD) that contained 187 children, including 92 ASD and 95 children typically developing (TD). Using computer vision techniques, some appearance features of children including facial appearing time, eye concentration analysis, response time to name calls, and emotional expression ability were extracted. Subsequently, these features were combined and machine learning methods were used for the classification of children. Notably, the Bayes classifier achieved a remarkable accuracy of 94.1%. The experimental results show that the extracted visual appearance features can reflect the typical symptoms of children, and the automatic recognition method can provide an auxiliary diagnosis or data support for doctors.

The ASD group were all pre-school children, aged between 20 and 60 months, with an average age of 33.4 months for males and 31.5 months for females.

Like it or not, it seems that autistic toddlers do look different and so it is not a hidden disability. Nobody should be waiting years for a diagnosis.

Bullying

Most autism diagnosed today is mild, level 1 autism. Some of this group really do struggle and can genuinely benefit from pharmacologic therapies.

Bullying is one very common issue that is faced and does not need drug therapy, it needs a different kind of intervention.

A preliminary analysis of teaching children with autism spectrum disorder self-protection skills for bullying situations

Children diagnosed with autism spectrum disorder are at high risk of being bullied, but research on teaching children with autism self-protection skills for bullying situations is scant. We taught five children self-protection skills for two types of bullying (threats and unkind remarks) and consecutive bullying occurrences. We first evaluated behavioral skills training and a textual prompt to teach children to report threats of physical or material harm, provide a disapproving statement after a first unkind remark, and occupy themselves with an activity away from a bully after a second unkind remark. Additional tactics were necessary to aid in the discrimination of bullying situations for two children. There were increases in the self-protection skills with all children. Results further support that an active-learning approach is efficacious in teaching responses to bullying in simulated situations. Considerations for teaching these skills while maintaining trust and rapport with children and caregivers are discussed.

Having a sibling in the same school can be an effective defence against bullying. It might be an older brother, as was the case for Monty, but a younger sister can also be very effective. One episode, of many, I witnessed at school was a young Swedish girl intervening on behalf of her older Aspie-like brother. It really shocked the older boys and certainty impressed me.

I think most bullying affects those with level 1 autism. Those with severe autism would tend to have a 1:1 assistant and if he/she is doing their job there should not be the possibility bullying. I am told that out in the real world kids with level 3 autism do get bullied, which means the system has failed.

From the school’s perspective there is also the opposite issue of the pupil with autism/ADHD attacking other pupils or staff. This does happen and if the child is a large fully-grown male can lead to very serious injury. It is not just those with level 3 autism who can do this.

I think the best strategy to protect against bullying is to ensure your child is in a caring environment at school and is well integrated. This may be easier said than done, but it is possible for many people. Then the other pupils will look out for the one with special needs. This assumes you do not overdo it with who gets to be "special".

Special needs are not so special any more, as was highlighted recently in the UK. For the most privileged group of pupils, those going to private fee-paying schools, 41% are getting special treatment in their exams due to their various special needs. Even in the regular state schools, which for sure have a higher percentage of kids with actual special needs, 26% of pupils get extra time in exams.

Nearly one in three pupils in England given extra time in exams, says regulator

Nearly a third of pupils in England were given 25% extra time to complete their GCSEs and A-level exams following a surge in special exam access arrangements being granted, data from Ofqual has shown.

The figure is higher again among exam candidates in private schools where more than two in five received 25% extra time in the last academic year, according to England’s exams regulator.

The total number of approved special access arrangements for GCSE, AS and A-level exams rose by 12.3% in the 2023/24 school year compared to the year before, the data has revealed.

·         Independent centres 41.8%

·         Sixth form and FE colleges 35%

·         Non-selective state schools 26.5%

It comes as education leaders have suggested more pupils are seeking support after the pandemic due to a rise in young people with special educational needs and disabilities (Send) and mental health issues.

Requests for 25% extra time in exams was the most common approved access arrangement for pupils with learning difficulties or disabilities, followed by computer readers, scribes and speech recognition.

 

Folate supplementation in mothers prevent pesticides causing neurodevelopmental disorders in offspring

There is a lot of research about folate (vitamin B9), birth defects and autism. From the early 1990s women were encouraged to take folate supplements during pregnancy to avoid neural tube defects and other congenital abnormalities.

Some individuals have mutations in the MTHFR gene that impair their ability to convert folic acid into its active form, L-methylfolate. For such individuals, taking methylated folate supplements will be necessary.

More recently we have learned that some people with adequate folate intake can lack folate inside their brain. They have antibodies that block the transmission of folate across the blood brain barrier.

We saw how one clinician is prescribing high dose calcium folinate to couples wishing to reduce the risk of autism in their future offspring, if they test positive themselves for folate receptor auto-antibodies.

As we already know exposure to pesticides and some other unnatural chemicals during pregnancy can lead to neurodevelopmental disorders (NDDs) that include autism.

The paper below is interesting because it looks as how to minimize the potential damage caused by exposure to pyrethroid pesticides, one of the most common classes of pesticides in the US.

Folate prevents the autism-related phenotype caused by developmental pyrethroid exposure in prairie voles 

Neurodevelopmental disorders (NDDs) have dramatically increased in prevalence to an alarming one in six children, and yet both causes and preventions remain elusive. Recent human epidemiology and animal studies have implicated developmental exposure to pyrethroid pesticides, one of the most common classes of pesticides in the US, as an environmental risk factor for autism and NDDs. Our previous research has shown that low-dose chronic developmental pyrethroid exposure (DPE) changes folate metabolites in the adult mouse brain. We hypothesize that DPE acts directly on molecular targets in the folate metabolism pathway, and that high-dose maternal folate supplementation can prevent or reduce the biobehavioral effects of DPE. We exposed pregnant prairie vole dams to vehicle or deltamethrin (3 mg/kg every 3 days) with or without folate supplementation (5 mg/kg methylfolate every 3 days). The resulting DPE offspring showed broad deficits in five behavioral domains relevant to NDDs; increased plasma folate concentrations; and increased neural expression of SHMT1, a cellular folate cycle enzyme. Maternal folate supplementation prevented most of the behavioral phenotype (except for repetitive behaviors) and caused potentially compensatory changes in neural expression of FOLR1 and MTHFR, two other folate-related proteins. We conclude that DPE causes NDD-relevant behavioral deficits; DPE directly alters aspects of folate metabolism; and preventative interventions targeting folate metabolism are effective in reducing, but not eliminating, the behavioral effects of DPE.

 

A round-up of therapies to treat mouse autism

Treating human autism is not yet mainstream, but treating autism in mice has been going on for decades. Of course the idea is to use mouse models with a view to later treating humans.

The paper below is about mice, but it is actually a very good summary of the current status of treatment options more broadly.

It even covers the use of HDAC inhibitors to use epigenetics as a treatment tool. Click on the link to read the full text for free. 

The Use of Nutraceutical and Pharmacological Strategies in Murine Models of Autism Spectrum Disorder 

Autism spectrum disorder (ASD) is a common neurodevelopmental condition mainly characterized by both a scarce aptitude for social interactions or communication and engagement in repetitive behaviors. These primary symptoms can manifest with variable severity and are often paired with a heterogeneous plethora of secondary complications, among which include anxiety, ADHD (attention deficit hyperactivity disorder), cognitive impairment, sleep disorders, sensory alterations, and gastrointestinal issues. So far, no treatment for the core symptoms of ASD has yielded satisfactory results in a clinical setting. Consequently, medical and psychological support for ASD patients has focused on improving quality of life and treating secondary complications. Despite no single cause being identified for the onset and development of ASD, many genetic mutations and risk factors, such as maternal age, fetal exposure to certain drugs, or infections have been linked to the disorder. In preclinical contexts, these correlations have acted as a valuable basis for the development of various murine models that have successfully mimicked ASD-like symptoms and complications. This review aims to summarize the findings of the extensive literature regarding the pharmacological and nutraceutical interventions that have been tested in the main animal models for ASD, and their effects on core symptoms and the anatomical, physiological, or molecular markers of the disorder.

The body of research here summarized suggests that many therapeutic strategies have yielded positive results for ASD core symptoms and ASD-linked cellular, anatomical, and metabolic alterations at the preclinical level. These results ultimately confirm clinical and in vitro evidence regarding the main pathways involved in ASD pathogenesis and hint at the potential for the combination of different types of treatment. The studies reviewed here showed that a treatment’s success or failure in these models usually depends on administration timing. The best results are commonly achieved when protective treatment is given in the first weeks after birth or prenatally. Unfortunately, this is not easily translatable into clinical practice as ASD diagnosis, at the moment, postdates this time window. Moreover, it is notable that most of the treatments employed in these studies did not achieve significant improvements in all the behavioral tests or definitive success in clinical trials. Despite the exact causes for the disparity between promising preclinical results and modest or negative clinical outcomes remaining unknown, a few hypotheses can be formulated. The results of many tests commonly employed to measure sociability and repetitive behaviors in mice can be altered by other symptoms known to be observed in these murine models, such as altered motor coordination, cognitive impairment, and anxiety, which may lead scientists to overestimate the effect of certain treatments on social behavior. Moreover, poor translatability may also be ascribed to the heterogeneity in symptoms and genetic backgrounds found in ASD human patients which, conversely, is far more limited in these mice strains. Ultimately, other possible confounding factors such as interactions with concurrent medications, socio-economic elements, patient lifestyle, or concomitant diseases are significantly more frequent and variable in the human population. Poor translatability may be potentially alleviated by precision medicine approaches in clinical practice and by preclinical testing of single treatments in a variety of ASD murine models. Ultimately, the present literature shows that, despite the limited clinical translational success, murine models can be a valuable tool for testing a variety of treatments in ASD research.

 

Figure 2. Schematic representation of key elements of the mTOR pathway and of therapeutic interventions considered in murine models for ASD. Abbreviations: PIP2: phosphatidylinositol 4,5-bisphosphate PIP3: phosphatidylinositol 3,4,5-bisphosphate PI3K: phosphatidylinositol 3-kinase; PTEN: phosphatase and tensin homolog; Akt: protein kinase B; TSC1: tuberous sclerosis 1; TSC2: tuberous sclerosis 2; AMPK: AMP-activated protein kinase; mTOR: mammalian target of rapamycin; mTORC1: mTOR complex 1; mTORC2: mTOR complex 2; S6K: Ribosomal protein S6 kinase beta-1; eIF4E: eukaryotic Initiation Factor 4E; ULK complex: Unc-51-like kinase 1 complex; PKCa: protein kinase C alpha; P: phosphate group

You can see all the amino acids that have been trialed to modify mTOR (taurine, lysine, histidine and threonine) plus metformin and the potent rapamycin.

Also mentioned is the WHEN in what I call the what, when and where in autism treatment. This is the idea of treatment windows, when a specific therapy can potentially be beneficial.

This very concept was discussed in a recent paper on Rett syndrome.

Protein Loss Triggers Molecular Changes Linked to Rett Syndrome 

Key Facts

·         Early Gene Changes: Loss of MeCP2 leads to immediate gene expression dysregulation, affecting hundreds of genes.

·         Neuronal Impact: Dysregulated genes are linked to neuronal function, causing downstream circuit-level deficits.

·         Therapeutic Window: The study reveals a time frame between molecular changes and neurological symptoms, enabling early intervention opportunities.

Another transcription factor (TCF) that causes autism

There is a lot in this blog about TCF4 (transcription factor 4). Loss of this gene leads to Pitt Hopkins syndrome. Disruption of the gene is associated with schizophrenia and intellectual disability.

Mutations in TCF20 lead to a kind of autism plus intellectual disability called TCF20-Related Neurodevelopmental Disorder. Like Pitt Hopkins, this is a rare disorder, but milder misexpression of the gene is likely much more common. In the recent paper below we see which are the downstream effector genes.

Our old friends the sub-units of GABAa receptors are there. In this case it is GABRA1 and GABRA5 that are reduced.

Both GABRA1 and GABRA5 play essential but distinct roles in regulating neuronal inhibition. GABRA1 primarily contributes to synaptic inhibition and is critical in seizure and anxiety regulation, while GABRA5 is involved in tonic inhibition and cognitive processes.

Malfunctions in GABRA1 and GABRA5 can lead to autism, anxiety, schizophrenia, intellectual disability, epilepsy etc

Regulation of Dendrite and Dendritic Spine Formation by TCF20

Mutations in the Transcription Factor 20 (TCF20) have been identified in patients with autism spectrum disorders (ASDs), intellectual disabilities (IDs), and other neurological issues. Recently, a new syndrome called TCF20-associated neurodevelopmental disorders (TAND) has been described, with specific clinical features. While TCF20's role in the neurogenesis of mouse embryos has been reported, little is known about its molecular function in neurons. In this study, we demonstrate that TCF20 is expressed in all analyzed brain regions in mice, and its expression increases during brain development but decreases in muscle tissue. Our findings suggest that TCF20 plays a central role in dendritic arborization and dendritic spine formation processes. RNA sequencing analysis revealed a downregulation of pre- and postsynaptic pathways in TCF20 knockdown neurons. We also found decreased levels of GABRA1, BDNF, PSD-95, and c-Fos in total homogenates and in synaptosomal preparations of knockdown TCF20 rat cortical cultures. Furthermore, synaptosomal preparations of knockdown TCF20 rat cortical cultures showed significant downregulation of GluN2B and GABRA5, while GluA2 was significantly upregulated. Overall, our data suggest that TCF20 plays an essential role in neuronal development and function by modulating the expression of proteins involved in dendrite and synapse formation and function.

Based on these results, we analyzed the expression of neuronal proteins in TCF20-deficient neurons and found decreased levels of GABRA1, BDNF, PSD-95, and c-Fos in total homogenates (Figure 5) and in synaptosomal preparations (Figure 5) of shTCF20 rat cortical cultures. Additionally, GluN2B and GABRA5 were significantly downregulated, and GluA2 was significantly upregulated in synaptosomal preparations of shTCF20 rat cortical cultures (Figure 5).

On the subject of GABA type A receptor, we have a very recent paper from Poland that delves into this subject in great detail. 

Molecular mechanisms of the GABA type A receptor function

The GABA type A receptor (GABA_AR) belongs to the family of pentameric ligand-gated ion channels and plays a key role in inhibition in adult mammalian brains. Dysfunction of this macromolecule may lead to epilepsy, anxiety disorders, autism, depression, and schizophrenia.

And finally …

Dr Frye has published a study that assessed the effect of his friend Dr Boles’ mitochondrial cocktail.

I did meet Dr Boles a while back at a conference in London. He came with his wife and a stock of NeuroNeeds products for sale, including SpectrumNeeds which was the subject of today’s paper. He was telling me all about the great food just across the border in Mexico and how he learnt Spanish.

A Mitochondrial Supplement Improves Function and Mitochondrial Activity in Autism: A double-blind placebo-controlled cross-over trial

Autism spectrum disorder (ASD) is associated with mitochondrial dysfunction but studies demonstrating the efficacy of treatments are scarce. We sought to determine whether a mitochondrial-targeted dietary supplement designed for children with ASD improved mitochondrial function and ASD symptomatology using a double-blind placebo-controlled cross-over design. Sixteen children [Mean Age 9y 4m; 88% male] with non-syndromic ASD and mitochondrial enzyme abnormalities, as measured by MitoSwab, received weight-adjusted SpectrumNeeds and QNeeds  and placebos matched on taste, texture and appearance during two separate 12-week blocks. Which product received first was randomized. The treatment significantly normalized citrate synthase and complex IV activity as measured by the MitoSwab. Mitochondrial respiration of peripheral blood mononuclear cell respiration, as measured by the Seahorse XFe96  with the mitochondrial oxidative stress test, became more resilient to oxidative stress after the treatment, particularly in children with poor neurodevelopment. The mitochondrial supplement demonstrated significant improvement in standardized parent-rated scales in neurodevelopment, social withdrawal, hyperactivity and caregiver strain with large effect sizes (Cohen’s d’ = 0.77-1.25), while changes measured by the clinical and psychometric instruments were not significantly different. Adverse effects were minimal. This small study on children with ASD and mitochondrial abnormalities demonstrates that a simple, well-tolerated mitochondrial-targeted dietary supplement can improve mitochondrial physiology, ASD symptoms and caregiver wellbeing. Further larger controlled studies need to verify and extend these findings. These findings are significant as children with ASD have few other effective treatments.

Conclusion

Plus ça change, plus c'est la même chose.

The more things change, the more they stay the same.

There isn’t much new that we don’t already know. This is probably good news.

I think for Dr Boles and our Spanish speaking readers you would say "Cuanto más cambian las cosas, más siguen igual." Correct me if I am wrong.

Nitrosative Stress, Nitric Oxide and Peroxynitrite

In this example of Brain Injury, developing oligodendrocytes are injured and killed by substances released from activated microglia, including nitric oxide and superoxide, which form peroxynitrite. Peroxynitrite has been found to kill these cells through the activation of the 12-lipoxygenase pathway for metabolizing arachidonic acid. Mitochondria may be involved in this pathway as a source of reactive oxygen species.

Much has been written in this blog about oxidative stress, which has now been extremely well researched in autism and more generally. Let’s recap oxidative stress.

The most knowledgeable researcher in this area is Abha Chauhan.  Based on her research and that of others we now know a great deal.  Recall that the body’s key antioxidant is called glutathione (GSH) and when it neutralizes a free radical GSH is converted to its oxidized form, glutathione disulfide (GSSG).  A good measure of oxidative stress is the ratio of  GSH/GSSG.

·        Autism is associated with deficits in glutathione antioxidant defence in selective regions of the brain.

·        In the cerebellum and temporal cortex from subjects with autism, GSH levels are significantly decreased by 34.2 and 44.6 %, with a concomitant increase in the levels of GSSG

·        There is also a significant decrease in the levels of total GSH (tGSH) by 32.9 % in the cerebellum, and by 43.1 % in the temporal cortex of subjects with autism.

·        In contrast, there was no significant change in GSH, GSSG and tGSH levels in the frontal, parietal and occipital cortices in autism

·        The redox ratio of GSH to GSSG was also significantly decreased by 52.8 % in the cerebellum and by 60.8 % in the temporal cortex of subjects with autism, suggesting glutathione redox imbalance in the brain of individuals with autism.

·        Disturbances in brain glutathione homeostasis may contribute to oxidative stress, immune dysfunction and apoptosis, particularly in the cerebellum and temporal lobe, and may lead to neurodevelopmental abnormalities in autism.

·        The activity of glutathione cysteine ligase (GCL), an enzyme for glutathione synthesis is impaired in autism.

·        The protein expression of its modulatory subunit GCLM was decreased in autism.

·        The activities of glutathione peroxidase (GPx) and glutathione S-transferase were decreased in autism.

For those interested, GPx is a family of enzymes that catalyze the reaction that converts GSH to GCCG.  So in order to soak up those free radicals you need both GSH and GPx.

Glutathione cysteine ligase (GCL) is a key enzyme needed to make the antioxidant GSH.  Dysregulation of GCL enzymatic function and activity is known to be involved in many human diseases, such as diabetes, Parkinson's disease, Alzheimer’s disease, COPD, HIV/AIDS, cancer and autism.  Without sufficient GCL your body cannot make enough glutathione (GSH).

I did have some conversation with Abha Chauhan a few years ago when I found that NAC (N-acetyl cysteine), a known precursor to GSH, really does have a positive behavioral impact in autism.  She is clearly very nice, but not the type to make the leap to translating her science into therapy.

As I have shown there are many other treatable aspects of oxidative stress.

The chart below is my annotated version of the original by Professor Helmut Sies, the German “Redox Pioneer”.  He has published 500 scientific papers.

Nitrosative Stress

Finally to nitrogen.

Nitrogen is the most common pure element in the earth, making up 78.1% of the entire volume of the atmosphere.  Although nitrogen is non-toxic, when released into an enclosed space it can displace oxygen, and therefore presents an asphyxiation hazard. 

Nitrogen is an anesthetic agent. Nitrous oxide (N₂O) is commonly known as laughing gas.  It is used in medicine for its unaesthetic and analgesic effects

It is also used as an oxidizer in rocket propellants, and in motor racing to increase the power output of engines, like Mad Max.

In humans we are dealing with Nitric Oxide (NO) and when things go wrong with peroxynitrite and then other Reactive nitrogen species (RNS).  In simple terms Reactive nitrogen species (RNS), like Reactive oxygen species (ROS) are bad news.

Nitric Oxide (NO) itself does lots of good things in your body.  Too much may not be good, but a little more can actually do you good.  NO is a potent vasodilator.

For over 130 years, nitroglycerin has been used to treat heart conditions, such as angina and chronic heart failure.  Nitroglycerin produces nitric oxide (NO). In hospital most patients will receive nitroglycerin during and after a heart attack, people at risk of a heart attack often carry nitroglycerin with them.

If you want to lower your blood pressure or an athlete wanting to legally improve exercise endurance you can increase Nitric Oxide (NO) via diet.  One easy way is to drink beetroot juice, as is common in endurance cycling.  In people with peroxynitrite-derived radicals this may be unwise, because they may have too much NO.

Peroxynitrite

The starting point for the production of those unhelpful Reactive Nitrogen Species (RNS) is this chemical reaction

NO (nitric oxide) + O₂^·− (superoxide) → ONOO⁻ (peroxynitrite)

NO production is affected by the enzyme nitric oxide synthase 2 (NOS2).

Superoxide production is catalyzed by NADPH oxidase.

Superoxide also produces Reactive Oxygen Species (ROS).

NADPH oxidase is implicated in many diseases including schizophrenia and autism.

NADPH oxidase 4 (Nox4) activity decreases mitochondrial function (chain complex I).

Activated microglia (as found in autism) produce both nitric oxide and superoxide and are therefore a source of peroxynitrite.

This has started to get rather complicated. So those interested in NADPH should refer to the literature.

Peroxynitrite can directly react with various biological targets and components of the cell including lipids, thiols, amino acid residues, DNA bases, and low-molecular weight antioxidants.

Additionally peroxynitrite can react with other molecules to form additional types of RNS including nitrogen dioxide (·NO₂) and dinitrogen trioxide (N₂O₃) as well as other types of chemically reactive free radicals.

Nitric Oxide and Peroxynitrite in Health and Disease

I have referred on this blog to Abha Chauhan’s mammoth book on oxidative stress in autism on several occasions.  A work of similar quality but this time on Nitric Oxide and Peroxynitrite, is the paper below, by Hungarian Pal Pacher, who works at the US National Institute of Health’s Section on Oxidative Stress Tissue Injury.  He looks like a citation generating machine.

You could spend a long time reading this paper, but in summary peroxynitrite and its derived products have a negative effect on a very wide range of conditions including all the common neurological conditions, inflammatory diseases and again diabetes.  The answer would be peroxynitrite scavengers.

Nitric Oxide and Peroxynitrite in Health and Disease

The discovery that mammalian cells have the ability to synthesize the free radical nitric oxide (NO) has stimulated an extraordinary impetus for scientific research in all the fields of biology and medicine. Since its early description as an endothelial-derived relaxing factor, NO has emerged as a fundamental signaling device regulating virtually every critical cellular function, as well as a potent mediator of cellular damage in a wide range of conditions. Recent evidence indicates that most of the cytotoxicity attributed to NO is rather due to peroxynitrite, produced from the diffusion-controlled reaction between NO and another free radical, the superoxide anion. Peroxynitrite interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms. These reactions trigger cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis. In vivo, peroxynitrite generation represents a crucial pathogenic mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, circulatory shock, chronic inflammatory diseases, cancer, and neurodegenerative disorders. Hence, novel pharmacological strategies aimed at removing peroxynitrite might represent powerful therapeutic tools in the future. Evidence supporting these novel roles of NO and peroxynitrite is presented in detail in this review.


Some excerpts:-

·        The different events set in motion by the initial generation of peroxynitrite indicate that potent peroxynitrite decomposition catalysts and PARP inhibitors might represent useful therapeutic agents for debilitating chronic inflammatory diseases

·        In summary, available evidence indicates that NO plays dichotomous roles (promotion vs. suppression) in tumor initiation and progression. The activation of angiogenesis and the induction of DNA mutations represent key aspects of the procarcinogenic effects of NO. Peroxynitrite is emerging as a major NO-derived species responsible for DNA damage, mainly through guanine modifications and the inhibition of DNA repair enzymes. In chronic inflammatory states, the identification of 8-nitroguanine in tissues indicates that nitrative DNA damage consecutive to overproduction of NO and peroxynitrite may represent an essential link between inflammation and carcinogenesis.

·        In summary, the different studies listed above indicate that small amounts of NO produced by eNOS in the vasculature during the early phase of brain ischemia are essential to limit the extent of cerebral damage, whereas higher concentrations of NO, generated initially by nNOS and later by iNOS, exert essentially neurotoxic effects in the ischemic brain. Evidence that such toxicity depends, in large part, on the rapid reaction of NO with locally produced superoxide to generate peroxynitrite will be now exposed

  

·        NO is produced by all brain cells including neurons, endothelial cells, and glial cells (astrocytes, oligodendrocytes, and microglia) by Ca²⁺/calmodulin-dependent NOS isoforms. Physiologically NOS in neurons (nNOS, type I NOS) and endothelial cells (eNOS, type III NOS) produce nanomolar amounts of NO for short periods in response to transient increases in intracellular Ca²⁺, which is essential for the control of cerebral blood flow and neurotransmission and is involved in synaptic plasticity, modulation of neuroendocrine functions, memory formation, and behavioral activity (491, 890, 1229). The brain produces more NO for signal transduction than the rest of the body combined, and its synthesis is induced by excitatory stimuli. Consequently, NO plays an important role in amplifying toxicity in the CNS. Indeed, under various pathological conditions associated with inflammation (e.g., neurodegenerative disorders and cerebral ischemia), large amounts of NO are produced in the brain as a result of the induced expression of iNOS (type II NOS) in glial cells, phagocytes, and vascular cells, which can exert various deleterious roles (39, 491, 890). Thus NO may be a double-edged sword, exerting protective effects by modulating numerous physiological processes and complex immunological functions in the CNS on one hand and on the other hand mediating tissue damage (446, 491, 890). The detailed discussion of the role of NO in the pathophysiology of various neurodegenerative disorders including Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS), just mentioning a few, is the subject of numerous excellent recent overviews (77, 145, 194, 219, 491, 890, 1003, 1205, 1433) and beyond the scope of this paper. Here we cover only the role of peroxynitrite and protein nitration, which are likely responsible for most deleterious effects of NO in neurodegenerative disorders.

·        Peroxynitrite formation has been implicated in Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, MS, ALS, and traumatic brain injury (reviewed in Refs. 194, 608, 1119, 1284). Nitrotyrosine immunoreactivity has been found in early stages of all of these diseases in human autopsy samples as well as in experimental animal models. Increased nitrite, nitrate, and free nitrotyrosine has been found to be present in the cerebral spinal fluid (CSF) and proposed to be useful marker of neurodegeneration (168; reviewed in Refs. 608, 1119, 1284). Once formed in the diseased brain, peroxynitrite may exert its toxic effects through multiple mechanisms, including lipid peroxidation, mitochondrial damage, protein nitration and oxidation, depletion of antioxidant reserves (especially glutathione), activation or inhibition of various signaling pathways, and DNA damage followed by the activation of the nuclear enzyme PARP (608, 1119, 1284).

·        Uric acid has proven to be a useful inhibitor of tyrosine nitration (although it is not a direct peroxynitrite scavenger) (1271) and has been shown to protect the blood-brain barrier and largely prevent the entry of inflammatory cells into the CNS (566, 567). Additionaly, it also prevented CNS injury after inflammatory cells have already migrated into the CNS (1141). Urate has also proven beneficial in reducing the morbidity associated with viral infections (710, 1141). Interestingly, in humans there is an inverse correlation between affliction with gout and MS (710, 1195). Numerous studies have reported lower levels of uric acid in MS patients favoring the view that reduced uric acid in MS is secondary to its “peroxynitrite scavenging” activity during inflammatory disease, rather than a primary deficiency (reviewed in Ref. 694). These studies have also suggested that serum uric acid levels could be used as biomarkers for monitoring disease activity in MS

  

·        Recent evidence suggests that mitochondrial complex I inhibition may be the central cause of sporadic PD and that derangements in complex I lead to α-synuclein aggregation, which contributes to the demise of dopamine neurons (293). Accumulation and aggregation of α-synuclein may further facilitate the death of dopamine neurons through impairments in protein handling and detoxification (293). As already mentioned above, both mitochondrial complex I and synuclein can be targets for peroxynitrite-induced protein nitration

·        The significance of this intricate interplay may have important ramifications not only for ALS but also for PD and AD (6, 58, 1102). Reactive astrocytes are common hallmark of neurodegeneration, and these results suggest that peroxynitrite may play an important role in promoting this phenotype as well as causing the degeneration of neurons. In ALS, the transformation of astrocytes into a reactive phenotype may explain why ALS is progressive, causing the relentless death of neighboring motor neurons. Interfering in such a cascade to stop the progressive death of motor neurons would not necessarily cure ALS but may keep it from being a death sentence.

·        There is accumulating evidence suggesting that increased oxidative stress and excessive production of NO might contribute to the development of HD by damaging neighboring neurons (reviewed in Refs. 63, 163). Accordingly, increased iNOS expression was observed in neuronal, glial, and vascular cells from brains of HD patients and mouse models of disease (206, 491). Similarly, numerous studies have demonstrated increased 3-NT formation in brain tissues (neurons, glia, and/or vasculature) of mice transgenic for the HD mutation, rats injected into the striatum with quinolinic acid (rat model of HD), and HD patients (300–302, 427, 1022, 1023, 1096, 1117). Importantly, both NOS inhibitors and peroxynitrite scavengers decreased neuronal damage, improved disease progression, and also decreased brain 3-NT content in experimental models (301, 1022, 1117). These results suggest that peroxynitrite might be an important mediator of oxidative damage associated with HD.

·        The pathogenetic role of peroxynitrite in TBI is supported by evidence demonstrating increased brain 3-NT levels following TBI in experimental mouse and rat models (92–94, 423, 507, 508, 898, 1171, 1360), and by the beneficial effects of NOS inhibitor and peroxynitrite scavengers in reducing neuronal injury and improving neurological recovery following injury (423, 508, 898).Collectively, multiple lines of evidence discussed above provide strong support for the important role of peroxynitrite formation and/or protein nitration in neurodegenerative disorders and suggest that the neutralization of this reactive species may offer significant therapeutic benefits in patients suffering from these devastating diseases.

·        Collectively, the evidence reviewed above support the view that peroxyntrite-induced damage plays an important role in numerous interconnected aspects of the pathogenesis of diabetes and diabetic complications. Neutralization of RNS or inhibition of downstream effector pathways including PARP activation may represent a promising strategy for the prevention or reversal of diabetic complications.

·        In conclusion, multiple lines of evidence discussed above and listed in Table 4 suggest that peroxynitrite plays an important role in various forms of cardiovascular dysfunction and injury; pharmacological neutralization of this reactive oxidant or targeting the downstream effector pathways may represent a promising strategy to treat various cardiovascular disorders.

·        In summary, circulatory shock is a leading cause of death in intensive care units. Considerable improvement in our understanding of the molecular and cellular mechanisms of shock over the past 20 years makes it now a reasonable expectation that novel, efficient mechanism-based therapies will emerge in the near future. Considerable evidence now exists that overproduction of NO and superoxide, triggering the generation of large amounts of peroxynitrite, is a central aspect of shock pathophysiology. In addition to direct cytotoxic effects such as the peroxidation of lipids, proteins, and DNA, peroxynitrite also occupies a critical position in a positive feedback loop of inflammatory injury, by (directly or indirectly, via PARP activation) activating proinflammatory signaling and by triggering the recruitment of phagocytes within injured tissues, leading to further NO, superoxide, and peroxynitrite production, which will progressively amplify the initial inflammatory reactions (see sect. VID, Fig. 14). These various observations support the view that future strategies reducing peroxynitrite or its precursors might have a considerable therapeutic impact in clinical circulatory shock.

Peroxynitrite Scavengers

We have already covered two substances in this blog that are potential Peroxynitrite Scavengers:-

Calcium Folinate

This is Roger’s magic pill to treat his Cerebral Folate Deficiency, but it may have application far beyond this, likely rare, condition, for those that tolerate it.

Tetrahydrofolic acid, or tetrahydrofolate, is a folic acid derivative. It has the potential to quench those peroxynitrite-derived radicals.

Tetrahydrofolate and 5-methyltetrahydrofolate are folates with high antioxidant activity. Identification of the antioxidant pharmacophore

The presumed protective effect of folic acid on the pathogenesis of cardiovascular, hematological and neurological diseases and cancer has been associated with the antioxidant activity of folic acid. Peroxynitrite (PON) scavenging activity and inhibition of lipid peroxidation (LPO) of the physiological forms of folate and of structurally related compounds were tested. It was found that the fully reduced forms of folate, i.e. tetrahydrofolate (THF) and 5-methyltetrahydrofolate (5-MTHF), had the most prominent antioxidant activity. It appeared that their protection against LPO is less pronounced than their PON scavenging activity. The antioxidant activity of these forms of folic acid resides in the pterin core, the antioxidant pharmacophore is 4-hydroxy-2,5,6-triaminopyrimidine. It is suggested that an electron donating effect of the 5-amino group is of major importance for the antioxidant activity of 4-hydroxy-2,5,6-triaminopyrimidine. A similar electron donating effect is probably important for the antioxidant activity of THF and 5-MTHF.

Uric Acid

Uric acid has proven to be a useful inhibitor of tyrosine nitration.  It was thought to be a scavenger of peroxynitrite, but our clever Pal from Hungary tells that “it is not a direct peroxynitrite scavenger ….Numerous studies have reported lower levels of uric acid in MS patients favoring the view that reduced uric acid in MS is secondary to its “peroxynitrite scavenging” activity during inflammatory disease, rather than a primary deficiency”.

An old paper:-

Uric acid, a natural scavenger of peroxynitrite, in experimental allergic encephalomyelitis and multiple sclerosis

Uric acid, the naturally occurring product of purine metabolism, is a strong peroxynitrite scavenger, as demonstrated by the capacity to bind peroxynitrite but not nitric oxide (NO) produced by lipopolysaccharide-stimulated cells of a mouse monocyte line. In this study, we used uric acid to treat experimental allergic encephalomyelitis (EAE) in the PLSJL strain of mice, which develop a chronic form of the disease with remissions and exacerbations. Uric acid administration was found to have strong therapeutic effects in a dose-dependent fashion. A regimen of four daily doses of 500 mg/kg uric acid was required to promote long-term survival regardless of whether treatment was initiated before or after the clinical symptoms of EAE had appeared. The requirement for multiple doses is likely to be caused by the rapid clearance of uric acid in mice which, unlike humans, metabolize uric acid a step further to allantoin. Uric acid treatment also was found to diminish clinical signs of a disease resembling EAE in interferon-γ receptor knockout mice. A possible association between multiple sclerosis (MS), the disease on which EAE is modeled, and uric acid is supported by the finding that patients with MS have significantly lower levels of serum uric acid than controls. In addition, statistical evaluation of more than 20 million patient records for the incidence of MS and gout (hyperuricemic) revealed that the two diseases are almost mutually exclusive, raising the possibility that hyperuricemia may protect against MS.

Here we have a paper with the link to Tetrahydrobiopterin (BH₄,), also known as sapropterin, covered in an old post:-

Endothelial Dysfunction - Oxidative Stress, Inflammation and BH4

Interactions of peroxynitrite with uric acid in the presence of ascorbate and thiols: Implications for uncoupling endothelial nitric oxide synthase

It has been suggested that uric acid acts as a peroxynitrite scavenger although it may also stimulate lipid peroxidation. To gain insight into how uric acid may act as an antioxidant, we used electron spin resonance to study the reaction of uric acid and plasma antioxidants with ONOO-. Peroxynitrite reacted with typical plasma concentrations of urate 16-fold faster than with ascorbate and 3-fold faster than cysteine. Xanthine but not other purine-analogs also reacted with peroxynitrite. The reaction between ONOO- and urate produced a carbon-centered free radical, which was inhibited by either ascorbate or cysteine. Moreover, scavenging of ONOO- by urate was significantly increased in the presence of ascorbate and cysteine. An important effect of ONOO- is oxidation of tetrahydrobiopterin, leading to uncoupling of nitric oxide synthase. The protection of eNOS function by urate, ascorbate and thiols in ONOO(-)-treated bovine aortic endothelial cells (BAECs) was, therefore, investigated by measuring superoxide and NO using the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine (CMH) and the NO-spin trap Fe[DETC]2. Peroxynitrite increased superoxide and decreased NO production by eNOS indicating eNOS uncoupling. Urate partially prevented this effect of ONOO- while treatment of BAECs with the combination of either urate with ascorbate or urate with cysteine completely prevented eNOS uncoupling caused by ONOO-. We conclude that the reducing and acidic properties of urate are important in effective scavenging of peroxynitrite and that cysteine and ascorbate markedly augment urate's antioxidant effect by reducing urate-derived radicals.

Xanthine oxidase (XO, sometimes 'XAO') is a form of xanthine oxidoreductase, a type of enzyme that generates reactive oxygen species.^[2] These enzymes catalyze the oxidation of hypoxanthine to xanthine and can further catalyze the oxidation of xanthine to uric acid. These enzymes play an important role in the catabolism of purines in some species, including humans.

Because xanthine oxidase is a metabolic pathway for uric acid formation, the xanthine oxidase inhibitor allopurinol is used in the treatment of gout.

Inhibition of xanthine oxidase has been proposed as a mechanism for improving cardiovascular health.  A study found that patients with chronic obstructive pulmonary disease (COPD) had a decrease in oxidative stress, including glutathione oxidation and lipid peroxidation, when xanthine oxidase was inhibited using allopurinol.

Reactive nitrogen species, such as peroxynitrite that xanthine oxidase can form, have been found to react with DNA, proteins, and cells, causing cellular damage or even toxicity. Reactive nitrogen signaling, coupled with reactive oxygen species, have been found to be a central part of myocardial and vascular function, explaining why xanthine oxidase is being researched for links to cardiovascular health.

We also should recall that abnormalities are common in autism.

The Hyperuricosuric Subtype of Autism, Uridine and Antipurinergic Therapy

So perhaps allopurinol for those with too much uric acid?  Perhaps this is a good marker for peroxynitrites ?

Conclusion

As is often the case there some contradiction in the science.  Is NO good for you or not?  Are both high and low uric acid actually indicating the same biological problem.

It looks like the research into very expensive BH4 therapy might be better directed into peroxynitrite scavengers.

I think we have found the reason why so many people with autism respond to Leucovorin (calcium folinate) and, unlike in our friend Roger, it may not be because of cerebral folate deficiency.

It looks like many other chronic conditions from diabetes to COPD to schizophrenia might also benefit from  calcium folinate.

Before I forget, in the Helmut Sies oxidative stress graphic I did highlight selenium.  The GPx enzymes contain selenium and if there is selenium deficiency the body's key antioxidant mechanism will be compromised. According to Abha Chauhan's book,  "Likewise, levels of exogenous antioxidants were also found to be reduced in autism, including vitamin C, vitamin E, and vitamin A in plasma, and zinc and selenium in erythrocytes (James et al., 2004)".  This might suggest adding a little extra selenium.

I think Allopurinol is worth a look for some autism.  Allopurinol does indeed reduce reactive nitrogen species in COPD (severe asthma), as suggested above.

Xanthine oxidase inhibition reduces reactive nitrogen species production in COPD airways

“These results suggest that oral administration of the xanthine oxidase inhibitor allopurinol reduces airway reactive nitrogen species production in chronic obstructive pulmonary disease subjects. This intervention may be useful in the future management of chronic "