Precisely controlling endogenous protein dosage in hPSCs and derivatives to model FOXG1 syndrome

Authors: Wenliang Zhu, Boya Zhang, Mengqi 
Journal: Nature Communications
Year: 2019

Dosage of key regulators impinge on developmental disorders such as FOXG1 syndrome. Since neither knock-out nor knock-down strategy assures flexible and precise protein abundance control, to study hypomorphic or haploinsufficiency expression remains challenging. We develop a system in human pluripotent stem cells (hPSCs) using CRISPR/Cas9 and SMASh technology, with which we can target endogenous proteins for precise dosage control in hPSCs and at multiple stages of neural differentiation. We also reveal FOXG1 dose-dependently affect the cellular constitution of human brain, with 60% mildly affect GABAergic interneuron development while 30% thresholds the production of MGE derived neurons. Abnormal interneuron differentiation accounts for various neurological defects such as epilepsy or seizures, which stimulates future innovative cures of FOXG1 syndrome. By means of its robustness and easiness, dosage-control of proteins in hPSCs and their derivatives will update the understanding and treatment of additional diseases caused by abnormal protein dosage.

A new study from State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, and Chinese Academy of Sciences, Beijing, China shows how SMASh enables tunable shut-off of transgene in hPSCs

Discovery of the genetic 'conductor' of brain stem cells

First Author: Antonello Mallamaci, SISSA 
Journal: Celebral Cortex
Year: 2019

Our brain comprises 85 billion nerve cells and just as many so-called glial cells, which work in close contact with the former to guarantee their proper function. All originate from brain stem cells. But what decides when and how many of them become neurons or glial cells? A new study led by the Laboratory of Cerebral Cortex Development of SISSA has shown how the Foxg1 gene, already involved in numerous processes of cerebral development and in rare disorders like Rett and West syndromes, plays a fundamental role in piloting the differentiation of stem cells, guaranteeing that neurons and glial cells are produced in the right quantity and at the right moment. The work, published in Cerebral Cortex and conducted in collaboration with the University of Cambridge and the IRCCS Burlo Garofolo, opens new roads to understanding and treating incurable genetic diseases.

A new study by SISSA identifies the gene which regulates the production ratio of neurons and support cells during cerebral cortex development..


First Author: Soo-Kyung Lee
Journal: Neuron
Year: 2018

The hallmarks of FOXG1 syndrome, which results from mutations in a single FOXG1 allele, include cortical atrophy and corpus callosum agenesis. However, the etiology for these structural deficits and the role of FOXG1 in cortical projection neurons remain unclear. Here we demonstrate that Foxg1 in pyramidal neurons plays essential roles in establishing cortical layers and the identity and axon trajectory of callosal projection neurons. The neuron-specific actions of Foxg1 are achieved by forming a transcription complex with Rp58. The Foxg1-Rp58 complex directly binds and represses Robo1, Slit3, and Reelingenes, the key regulators of callosal axon guidance and neuronal migration. We also found that inactivation of one Foxg1allele specifically in cortical neurons was sufficient to cause cerebral cortical hypoplasia and corpus callosum agenesis.

Study reveals a novel gene regulatory pathway that specifies neuronal characteristics during cerebral cortex development and sheds light on the etiology of FOXG1 syndrome.

Delineating FOXG1 syndrome From congenital microcephaly to hyperkinetic encephalopathy

First Author: Nancy Vegas, MD
Journal: Neurology Genetics
Year: 2018

A new case series led by Dr. Bahi-Buisson adds 45 additional ‘foxes’ to the medical literature (bringing the total number >130). Importantly, this study increases the range of ages reported in FOXG1 syndrome - 6 of the new cases are 18 years or older, providing valuable information about what life is like for affected adults. As seen in other case series, the majority of causative mutations are ‘frameshift’ variants that remove part of the FOXG1 protein.

The first symptoms most commonly reported in the 45 new cases included: developmental delay, microcephaly (small head size), and differences in visual tracking. By childhood, FOXG1 syndrome is characterized by: developmental and intellectual disabilities, limited functional hand use, and a complex movement disorder. Seizures are reported in ~78%, with age of onset ranging from shortly after birth to childhood; in about half, seizures are not controlled by medication. An important part of this case series focused on brain imaging findings in people with FOXG1 syndrome. The authors describe three main abnormalities: (1) delayed myelination, (2) abnormal gyri, and (3) abnormal corpus callosum. Myelination, which continues even after birth, is an important process that helps neurons transmit information. Brain imaging in FOXG1 syndrome shows that this process may occur more slowly. The gyri are the folds of the brain; in some people with FOXG1 syndrome, there can be fewer folds in the front part of the brain. The corpus callosum is a large tract that connects the two hemispheres of the brain. In FOXG1 syndrome, this tract can look smaller or may be absent. Importantly, in a small number of cases who had brain imaging earlier and later in life, there is interval development. Finally, information from this study supports other work that suggests mutations towards the beginning of the FOXG1 gene, or complete absence of the FOXG1 gene, may be associated with more severe symptoms.

Significance As more cases are published in the medical literature, our understanding of FOXG1 syndrome grows. Knowing the number of people with FOXG1 syndrome and identifying clinical endpoints are important tools for developing treatments.

Phenotype differentiation of FOXG1 and MECP2 disorders: a new method for characterization of developmental encephalopathies

First Author: Mandy Ma
Journal: The Journal of Pediatrics
Year: 2016

Developed the ‘Developmental Encephalopathy Inventory’, a tool to measure and differentiate between different features of individuals with autism spectrum disorders or intellectual disabilities. The DEI is designed to evaluate fourteen different domains, including: ambulation, fine motor skills, language, mood, movements, seizures, sensory functions, etc. The authors used the DEI to compare features of individuals with FOXG1 syndrome and Rett syndrome. For both disorders, fine motor and expressive language skills were often impaired. The DEI was able to identify features that distinguished the two syndromes; generally, FOXG1 syndrome was assessed as being more severe, whereas those with Rett syndrome tended to become more severely affected with age.

Developed a systematic way to evaluate clinical severity in individuals with FOXG1 syndrome. This can be used to measure the success or limitations of proposed therapies in clinical trials.

The hyperkinetic movement disorder of FOXG1-related epileptic-dyskinetic encephalopathy

First Author: Elena Cellini
Journal: Developmental Medicine & Child Neurology
Year: 2016

The authors characterized video recordings of movement disorders in 8 children with FOXG1 syndrome. The observed movement disorder was described as ‘complex’, with combinations of dyskinetic and hyperkinetic movements, like
dystonia, chorea, and athetosis.

Provides improved characterization of the movement disorders in individuals with FOXG1 syndrome, which may be important for medical management.

FOXG1 syndrome: genotype-phenotype association in 83 patients with FOXG1 variants

First Author: Diana Mitter
Journal: Genetics in Medicine
Year: 2016

The authors compared the clinical features of 83 individuals to determine whether any patterns could be identified based on the types of observed pathogenic variants (frameshift, missense, nonsense).

In general, those individuals with truncating variants in the N-terminal (beginning) domain of FOXG1 had a more severe presentation. Individuals with pathogenic missense variants in an evolutionarily conserved region of the forkhead
DNA-binding domain had milder presentations. This article also reports if and when individuals achieved developmental milestones: 45% were able to sit unassisted at a mean age of 28 months; 15% were able to walk unsupported at a mean age
of 53 months; functional hand use observed in 40%; 21% had some verbal expression (note that age at last follow-up was not consistent, and different for each individual).

A range of epilepsy diagnoses were reported, with a wide range in the age of onset (3 to 168 months). There does not seem to be a characteristic seizure type.

Importantly, 5 families have now been documented with presumed parental mosaicism; the authors recommend that prenatal genetic diagnosis be offered to families with a child who has FOXG1 syndrome.

First publication demonstrating genotype-phenotype correlations (i.e. the type of FOXG1 mutation impacts clinical severity), and uses a new standardized assessment of clinical severity in the largest published population of individuals with FOXG1 syndrome.

The role of FOXG1 in brain development and function

FOXG1-dependent dysregulation of GABA/Glutamatergic neuron differentiation in autism spectrum disorders

First Author: Jessica Mariani
Journal: Cell
Year: 2015

iPSC neurons generated from families with a proband dx with idiopathic ASD and increased head circumference; FOXG1 expression consistently shown to be upregulated in these individuals.

shRNA designed to target and downregulate FOXG1 expression; when ASD-derived iPSC cells treated, FOXG1 expression levels lowered to a level similar to their unaffected controls, with downregulation of GABAergic markers, with restoration to the normal level of GABAergic neuronal differentiation. Evidence also suggest upregulation of FOXG1 in ASD neural cells is an early driving force for proliferation of npcs of GABAergic lineage.

In ASD, larger head size has been associated with more severe symptoms and lower IQ; in 9 individuals with ASD and a larger head size; increased FOXG1 expression correlated with greater head size.

Implicates FOXG1 dysregulation in the pathogenesis of autism spectrum disorders, and possible in head size.

Imbalance of excitatory/inhibitory synaptic protein expression in iPSC-derived neurons from FOXG1 +/- patients and in Foxg1 +/- mice

First Author: Tommaso Patriarchi
Journal: European Journal of Human Genetics
Year: 2016

iPSC neurons generated from fibroblasts of two individuals with FOXG1 syndrome, one carrying a truncating variant, and the other with a deletion including the entire FOXG1 gene.

Similar to what is found in iPSC neurons from individuals with CDKL5 or MECP2-related disorders, expression of the gene, GRID1, was significantly increased in FOXG1-derived neurons. To compare the relative amounts of excitatory vs. inhibitory synapses, the expression of related gene markers were analyzed; those associated with inhibitory synapses were increased, whereas those with excitatory synapses were decreased. The authors propose that this imbalance may be contributing to the neurodevelopmental features observed in FOXG1 syndrome

First published use of iPSC neurons derived from individuals with FOXG1 syndrome; a phenotype is identified, which can be used as a marker in future experiments to measure the success of potential therapies.

Winged helix transcription factor BF-1 is essential for the development of the cerebral hemispheres

First Author: Shouhong Xuan
Journal: Neuron
Year: 1995

A mouse lacking Foxg1 expression was generated by replacing the coding sequencing with a lacZ/neomycin cassette; heterozygotes (only one copy of Foxg1 absent) were fertile and indistinguishable from wild-type littermates. Homozygotes were live born, but flaccid, with minimal spontaneous movements, and died shortly after birth.

Homozygotes were noted to have a significant reduction in brain size. The ventral telencephalon was noted to be more severely affected than the dorsal telencephalon; almost all telencephalon examined at E12.5 expressed dorsal markers, and not ventral markers. The authors further demonstrate that at E10.5, only dorsal telencephalic neuroepithelial cells are actively proliferating – ventral telencephalon cells are not. Authors suggest that their data demonstrate that absence of Foxg1 results in early withdrawal of telencephalic NPCs from the cell cycle, and premature onset of neuronal differentiation.

First Foxg1 animal model published; associated differences in brain development well-documented in the homozygous state.

RNA activation of haploinsufficient Foxg1 gene in murine neocortex

First Author: Cristina Fimiani
Journal: Scientific Reports
Year: 2016

Foxg1-RNAa delivered to murine E12.5 neocortical precursors by lentivirus; expression increase ranged from 1.28-2.88 fold increase. Increasing Foxg1expression in proliferating murine neocortical precursors decreased the number of neurons generated.

By delivering the RNAa molecule to specific neuronal populations, the authors provided evidence to suggest that ectopic (or off-target) up-regulation of Foxg1expression would not be significant.

Delivery of a similar miR molecule using an AAV9 system into newly born mouse pups showed modest upregulation of Foxg1 expression in the brain.

Demonstrates efficacy of using RNA activation to boost endogenous expression of Foxg1; this is a potential therapeutic strategy to alleviate clinical symptoms of FOXG1 syndrome