This White Paper, written by renowned scientists, is aimed at all stakeholders, all societies, their government and their media, for a global awareness. It will widely disseminate valuable knowledge for a coherent and rational public action, of interest for the public health and also of interest for the improvement of the human condition of the autistic people, without differences or borders.
Why have we failed to develop novel therapeutic avenues to developmental brain disorders?
Par Yehezkel Ben-Ari
The importance of autism in the public has led to repeated governmental plans aimed at understanding and treating that disorder and providing better care to children suffering from Autism Spectrum Disorders (ASD). Yet, these programs –at least in the domain of basic science that is discussed below- fail or have limited impact. I discuss below the reasons for these failures.
The genetic and molecular extraordinary achievements of the last decades have led to an impressive accumulation of data, identification of novel mutations and various intracellular cascades presumed to play a role in the pathogenesis of Developmental Brain Disorders (DBD). Yet, in spite of this, there is no novel treatment for Autism Spectrum Disorders (ASD), Rett syndrome, developmental epilepsies, and the plethora of genetic or environmental conditions due infantile disorders with life- long deleterious sequels.
There are 3 potential reasons for that:
i) Genetic or environmental Intrauterine insults deviate developmental sequences and this deviation rather than the insult per se is the main cause of the disorder. Indeed, Ben-Ari and Spitzer (2010) have suggested that neuronal activity and genetic programs act in series, the former validating the correct implementation of the latte Deviations of the developmental sequence of ionic currents or network patterns produced by a genetic mutation and/or an environmental insult in utero lead to a persistence of immature currents and/or networks in the mature nervous system (including the excitatory GABA). According to this “neuro-archeology” concept (Ben-Ari, 2008), neurons that fail to perform their assigned tasks and are misplaced or misconnected generate in the adult brain immature currents that perturb the operation of brain networks constituting pre-symptomatic electrical and/or architectural signatures of the disorder. This concept has been confirmed in many genetic migration disorders notably the double cortex mutation and mutations associated with seizures and mental disorders (Ackman et al., 2009). If so then the identification of the immature features (notably currents) offers the best approach to develop novel drugs as these will act on the “immature pathological features” ignoring the other normal operating neurons.
ii) Paucity of non-genetic investigations
The domination of the genocentric approach has fueled most funds and energy in the direction of identifying novel mutations and intracellular cascades hampering studies on the electrophysiological, morphological and biochemical studies of the pathogenic brains. This is particularly striking concerning the developing intrauterine or early post-natal that has literally been ignored in almost all investigations until very recently. Studies aimed at understanding ASD or Rett or Fragile X are almost exclusively performed in adolescents or adult brains in spite of the compelling evidence that the disorder starts in utero or during delivery. Intrauterine or delivery insults associated for instance with preterm delivery and in utero inflammations are ignored from basic science that is dedicated to understand & treat ASD and related disorders.
iii) Artificial separation of basic science and translational research
Developing novel drugs can only stem from a deep understanding of the operation of brain networks and neurons in health and disease. There is no applied science; there are applications of basic science. The two approaches are completely convergent and must be performed hand in hand and in close proximity. It is essential that the researchers trying to understand and treat pathologies remain close to those trying to understand how it operates in normal conditions. The determination of basic processes at work then allows then understanding how this is deviated in the pathogenic processes and how to correct that deviation.
In sum, there is a fundamental need to shift from largely dominated genetic research aimed at finding new mutations to a physiological approach centered on intrauterine life and delivery in order to understand how the brain matures in health and disease. This includes imaging, network investigations, use of genetic mice dedicated to label neurons and mapping the entire brain early on in health and disease.
Autism spectrum disorders (ASDs) and Fragile X are developmental disorders (DDs) manifested with diverse phenotypical outcomes including poor communication, aberrant social interactions, agitations, etc. (Levy, Mandell & Schultz, 2009; Fountain, Winter & Bearman, 2012; Abrahams & Geschwind, 2008; Betancur, 2011). It is estimated that approximately 1 in 100 children display signs and symptoms that lead to ASD (Fombonne, Quirke & Hagen, 2009; Weintraub, 2011) making it more common than childhood cancer and juvenile diabetes together. The most related genetic disorders investigated are Tuberous Sclerosis (TSC), Rett, Fragile X, Prader-Willi all endowed with ASD features (Khwaja & Sahin, 2011; Neul et al., 2010), and many events linked to pregnancy and delivery conditions. Down syndrome (DS) is included here because of recent observations suggesting common mechanisms and possible novel treatments. There is at present no FDA or EMA approved treatment for any of these disorders. Experimental studies have been used to advance our knowledge through a range of approaches at the genetic, molecular, cellular, synaptic, local circuit, circuit, systems, and behavioral levels (Bourgeron, 2009; Schmeisser et al., 2012; Spooren, Lindemann, Ghosh & Santarelli, 2012).
Genetic disorders
Tuberous Sclerosis (TSC) is caused by dominant mutations in either TSC1 or TSC2 tumour suppressor genes is characterized by the presence of brain malformations, the cortical tubers that are thought to contribute to the generation of pharmaco-resistant epilepsy but also to ASD features and other neurological disorders. We have recently discovered aberrant features of NMDA mediated currents in animal models and human resected slices (Lozovaya et al., 2014). Blocking these aberrant subunits selectively also blocked the seizures in both preparations.
Rett Syndrome (RTT) is an X-linked neurodevelopmental disorder primarily affecting girls. Over 95% of typical RTT cases are due to mutations in the gene encoding the transcriptional modulator methyl- CpG-binding protein 2 (MECP2)(Amir et al., 1999). RTT children have apparently normal early psychomotor development followed by profound developmental stagnation and regression after the age of 6 months, with loss of fine motor skills and language, and acquisition of hand stereotypies and autistic behavior, however, it is now clear that developmental processes are involved with earlier clinical manifestations. Indeed, RTT is associated with a plethora of other deleterious manifestations including seizures, respiratory dysfunction, gastrointestinal disease, anxiety, sleep disorders, autonomic abnormalities and growth failure, mood disorders, scoliosis, dystonia and Parkinsonism leading to progressive psychomotor deterioration (Neul et al., 2010; Armstrong, 2005; Percy et al., 2010). Neuro-pathological studies demonstrate lower brain weights and smaller, densely packed neurons but no evidence of degeneration or atrophy (Armstrong, 2005). Experimental observations reinforce the notion of a Parkinson type damage in striatal neurons in Rett.
Prader-Willi Syndrome (PWS) is a genetic disorder associated with lack of food intake at birth mediated by oxytocin signals and subsequent severe metabolic and behavioral manifestations including mental retardation and autism (Cassidy & Driscoll, 2008). PWS mice die at birth unless force-fed, but a single administration of oxytocin rescued the pups (Schaller, Watrin, Sturny, Massacrier, Szepetowski & Muscatelli, 2010). Similar observations have now been observed in babies with (Tauber et al., 2011).
Additional major pathological sources that are also major issues of public health: Preterm delivery, C-sections, complications during labor and birth and Developmental Disorders.
Mammalian delivery is one of the most complex biological processes, yet the alterations of electrical activity that occur during the process had not been investigated. The emergence of life from the sea to terrestrial environment in mammals that took millions of years during evolution is accomplished within hours through labor and delivery. In their seminal paper “The ‘Stress’ of Being Born” (Lagercrantz & Slotkin, 1986), the authors emphasized 4 important transitions during delivery: a shift from aquatic to dry environment with oxygen acquired through the lungs instead of the placenta, a reduction of environment temperature, a replacement of continuous to transient nutrient supply and a shift from a sterile bacterial environment to a neonatal microbiota that plays an important role in the immune system that matures during delivery (Romero & Korzeniewski, 2013; Hsiao et al., 2013). A series of complex and important mechanisms underlie the clearance of fetal lung fluid, surfactant secretion, and breathing, transition of fetal to neonatal circulation, decrease in pulmonary vascular resistance and pulmonary blood flow. The squeezing of the fetus through the birth canal and the outside cold temperature trigger an enormous surge of norepinephrine and epinephrine levels rising within minutes of term delivery and cord clamping, to levels never observed subsequently even after severe stress (Faxelius, Hägnevik, Lagercrantz, Lundell & Irestedt, 1983; Lagercrantz & Bistoletti, 1977). It is important to determine whether and how are these processes altered by intrauterine insults that will lead to autism and other Developmental disorders. In this aim it is instrumental to compare the alterations occurring shortly before and after birth in animal models of ASD and other disorders.
Delivery is a critic al period that attenuates or aggravates intrauterine insults (Ben-Ari, 2015) and oxytocin is thought to play an important role. Thus, low oxytocin signals are associated with poorer parent/child communication, and in rodents knock out of oxytocin signals are associated with autistic features (Sala et al., 2013). The incidence of autism is increased by complications during delivery, hypertension, pre-eclampsia, anoxic episodes and preterm delivery (Johnson, Hollis, Kochhar, Hennessy, Wolke & Marlow, 2010; Glasson, Bower, Petterson, de Klerk, Chaney & Hallmayer, 2004; Gardener, Spiegelman & Buka, 2009). Epidemiological investigations on the links between autism and C-sections have led to controversial results most likely because of the need to better take into account the various types of C-sections, but a recent meta-analysis confirms this link (Curran, O’Neill, Cryan, Kenny, Dinan, Kashan & Kearney, 2015). Delivery in rodent is associated with an abrupt oxytocin mediated neuroprotective excitatory to inhibitory shift of GABA actions –mediated by a reduction of intracellular chloride (Tyzio et al., 2006). Oxytocin receptor antagonists abolish this shift leading to excessive synchronization and hyperactivity (Tyzio et al., 2006; Tyzio et al., 2014) that might perturb social communication during a crucial moment. Therefore, early aberrant activities and paucity of oxytocin signals during delivery might produce long-term deleterious sequels. Interestingly, in both a drug induced animal model of ASD and in Fragile X mice, maternal administration of a diuretic that reduces intracellular chloride levels thereby restoring GABAergic inhibition during this crucial moment also attenuates the severity of ASD in offspring confirming the importance of labor and delivery in the pathogenesis of ASD (Tyzio et al., 2014; He, Nomura, Xu & Contractor, 2014). Collectively, these studies stress the need for more intrauterine and early post- natal period studies aimed at determining the developmental mechanisms underlying the pathogenesis of ASD.
Maternal Immune Activation (MIA).
Dys-regulation of the immune system has been extensively observed in ASD (Hsiao et al., 2013; Onore, Careaga & Ashwood, 2012; Hsiao, McBride, Chow, Mazmanian & Patterson, 2012; Shi, Smith, Malkova, Tse, Su & Patterson, 2009; Malkova, Yu, Hsiao, Moore & Patterson, 2012). Recent studies suggest that MIA disrupts microbiota in addition to the immune system in offspring and both these pathogenic events perturb the construction of the neocortex that is the final cause of the disease as correcting these insults also restores the correct cortical organization and attenuates the severity of the syndrome (Kim et al., 2017; Choi et al., 2016, Shin Yim et al., 2017). Because of this phenotypic overlap, ASDs and other DDs are potentially diseases involving the gut and the immune systems in addition to the nervous system, and linked to the latter via inflammation molecules.
Clinical trials relying on these observations
If neurons with immature features are present in ASD and perturb the operation of networks, then drugs blocking these activities might provide a sort of pharmaceutical surgery attenuating these features selectively. Indeed, relying on the neuro-archeology concept (Ben-Ari, 2008), we have tested the clinical efficacy of bumetanide a drug that restores GABAergic inhibition and obtained promising results in 2 large trials (Lemonnier et al., 2012; Lemonnier et al., 2017). A final European phase 3 trial will start soon (400 children -2 to 18 years old). Eye tracking and brain imaging open trials confirm the amelioration of visual contact by bumetanide (Hadjikhani et al., 2015; Hadjikhani et al., 2018).
In conclusion, these observations collectively stress the need for more developmental studies on the intrauterine, delivery and early post-natal period in order to better understand the pathogenesis of ASD. In parallel, the use of drugs capable of blocking selectively immature currents might provide a novel therapeutic perspective to treat ASD and other Developmental disorders. The aims here are to reduce as early as possible the perturbing signals as we know that early interventions are all the more efficient as they reduce the insults related to pathogenic activities during a crucial period (Wallace & Rogers, 2010; Dawson et al., 2010).
