Kimberly A. Aldinger

Consensus Paper: Pathological Role of the Cerebellum in Autism

There has been significant advancement in various aspects of scientific knowledge concerning the role of cerebellum in the etiopathogenesis of autism. In the current consensus paper, we will observe the diversity of opinions regarding the involvement of this important site in the pathology of autism. Recent emergent findings in literature related to cerebellar involvement in autism are discussed, including: cerebellar pathology, cerebellar imaging and symptom expression in autism, cerebellar genetics, cerebellar immune function, oxidative stress and mitochondrial dysfunction, GABAergic and glutamatergic systems, cholinergic, dopaminergic, serotonergic, and oxytocin-related changes in autism, motor control and cognitive deficits, cerebellar coordination of movements and cognition, gene–environment interactions, therapeutics in autism, and relevant animal models of autism. Points of consensus include presence of abnormal cerebellar anatomy, abnormal neurotransmitter systems, oxidative stress, cerebellar motor and cognitive deficits, and neuroinflammation in subjects with autism. Undefined areas or areas requiring further investigation include lack of treatment options for core symptoms of autism, vermal hypoplasia, and other vermal abnormalities as a consistent feature of autism, mechanisms underlying cerebellar contributions to cognition, and unknown mechanisms underlying neuroinflammation.

FOXC1 is required for normal cerebellar development and is a major contributor to chromosome 6p25.3 Dandy-Walker malformation

Dandy-Walker malformation (DWM), the most common human cerebellar malformation, has only one characterized associated locus. Here we characterize a second DWM-linked locus on 6p25.3, showing that deletions or duplications encompassing FOXC1 are associated with cerebellar and posterior fossa malformations including cerebellar vermis hypoplasia (CVH), mega-cisterna magna (MCM) and DWM. Foxc1-null mice have embryonic abnormalities of the rhombic lip due to loss of mesenchyme-secreted signaling molecules with subsequent loss of Atoh1 expression in vermis. Foxc1 homozygous hypomorphs have CVH with medial fusion and foliation defects. Human FOXC1 heterozygous mutations are known to affect eye development, causing a spectrum of glaucoma-associated anomalies (Axenfeld-Rieger syndrome, ARS; MIM no. 601631). We report the first brain imaging data from humans with FOXC1 mutations and show that these individuals also have CVH. We conclude that alteration of FOXC1 function alone causes CVH and contributes to MCM and DWM. Our results highlight a previously unrecognized role for mesenchyme-neuroepithelium interactions in the mid-hindbrain during early embryogenesis.

Genetic variation and population substructure in outbred CD-1 mice: implications for genome-wide association studies.

Outbred laboratory mouse populations are widely used in biomedical research. Since little is known about the degree of genetic variation present in these populations, they are not widely used for genetic studies. Commercially available outbred CD-1 mice are drawn from an extremely large breeding population that has accumulated many recombination events, which is desirable for genome-wide association studies. We therefore examined the degree of genome-wide variation within CD-1 mice to investigate their suitability for genetic studies. The CD-1 mouse genome displays patterns of linkage disequilibrium and heterogeneity similar to wild-caught mice. Population substructure and phenotypic differences were observed among CD-1 mice obtained from different breeding facilities. Differences in genetic variation among CD-1 mice from distinct facilities were similar to genetic differences detected between closely related human populations, consistent with a founder effect. This first large-scale genetic analysis of the outbred CD-1 mouse strain provides important considerations for the design and analysis of genetic studies in CD-1 mice.

Association and Mutation Analyses of 16p11.2 Autism Candidate Genes

Background Autism is a complex childhood neurodevelopmental disorder with a strong genetic basis. Microdeletion or duplication of a ∼500–700-kb genomic rearrangement on 16p11.2 that contains 24 genes represents the second most frequent chromosomal disorder associated with autism. The role of common and rare 16p11.2 sequence variants in autism etiology is unknown. Methodology/Principal Findings To identify common 16p11.2 variants with a potential role in autism, we performed association studies using existing data generated from three microarray platforms: Affymetrix 5.0 (777 families), Illumina 550 K (943 families), and Affymetrix 500 K (60 families). No common variants were identified that were significantly associated with autism. To look for rare variants, we performed resequencing of coding and promoter regions for eight candidate genes selected based on their known expression patterns and functions. In total, we identified 26 novel variants in autism: 13 exonic (nine non-synonymous, three synonymous, and one untranslated region) and 13 promoter variants. We found a significant association between autism and a coding variant in the seizure-related gene SEZ6L2 (12/1106 autism vs. 3/1161 controls; p = 0.018). Sez6l2 expression in mouse embryos was restricted to the spinal cord and brain. SEZ6L2 expression in human fetal brain was highest in post-mitotic cortical layers, hippocampus, amygdala, and thalamus. Association analysis of SEZ6L2 in an independent sample set failed to replicate our initial findings. Conclusions/Significance We have identified sequence variation in at least one candidate gene in 16p11.2 that may represent a novel genetic risk factor for autism. However, further studies are required to substantiate these preliminary findings.

Lmx1a regulates fates and location of cells originating from the cerebellar rhombic lip and telencephalic cortical hem

The cerebellar rhombic lip and telencephalic cortical hem are dorsally located germinal zones which contribute substantially to neuronal diversity in the CNS, but the mechanisms that drive neurogenesis within these zones are ill defined. Using genetic fate mapping in wild-type and Lmx1a−/− mice, we demonstrate that Lmx1a is a critical regulator of cell-fate decisions within both these germinal zones. In the developing cerebellum, Lmx1a is expressed in the roof plate, where it is required to segregate the roof plate lineage from neuronal rhombic lip derivatives. In addition, Lmx1a is expressed in a subset of rhombic lip progenitors which produce granule cells that are predominantly restricted to the cerebellar posterior vermis. In the absence of Lmx1a, these cells precociously exit the rhombic lip and overmigrate into the anterior vermis. This overmigration is associated with premature regression of the rhombic lip and posterior vermis hypoplasia in Lmx1a−/− mice. These data reveal molecular organization of the cerebellar rhombic lip and introduce Lmx1a as an important regulator of rhombic lip cell-fate decisions, which are critical for maintenance of the entire rhombic lip and normal cerebellar morphogenesis. In the developing telencephalon Lmx1a is expressed in the cortical hem, and in its absence cortical hem progenitors contribute excessively to the adjacent hippocampus instead of producing Cajal-Retzius neurons. Thus, Lmx1a activity is critical for proper production of cells originating from both the cerebellar rhombic lip and the telencephalic cortical hem.

Mutations in LAMA1 Cause Cerebellar Dysplasia and Cysts with and without Retinal Dystrophy

Cerebellar dysplasia with cysts (CDC) is an imaging finding typically seen in combination with cobblestone cortex and congenital muscular dystrophy in individuals with dystroglycanopathies. More recently, CDC was reported in seven children without neuromuscular involvement (Poretti-Boltshauser syndrome). Using a combination of homozygosity mapping and whole-exome sequencing, we identified biallelic mutations in LAMA1 as the cause of CDC in seven affected individuals (from five families) independent from those included in the phenotypic description of Poretti-Boltshauser syndrome. Most of these individuals also have high myopia, and some have retinal dystrophy and patchy increased T2-weighted fluid-attenuated inversion recovery (T2/FLAIR) signal in cortical white matter. In one additional family, we identified two siblings who have truncating LAMA1 mutations in combination with retinal dystrophy and mild cerebellar dysplasia without cysts, indicating that cysts are not an obligate feature associated with loss of LAMA1 function. This work expands the phenotypic spectrum associated with the lamininopathy disorders and highlights the tissue-specific roles played by different laminin-encoding genes.

Cerebellar and posterior fossa malformations in patients with autism-associated chromosome 22q13 terminal deletion.

The 22q13.3 deletion causes a neurodevelopmental syndrome, also known as Phelan‐McDermid syndrome (MIM #606232), characterized by developmental delay and severe delay or absence of expressive speech. Two patients with hemizygous chromosome 22q13.3 telomeric deletion were referred to us when brain‐imaging studies revealed cerebellar vermis hypoplasia (CBVH). To determine whether developmental abnormalities of the cerebellum are a consistent feature of the 22q13.3 deletion syndrome, we examined brain‐imaging studies for 10 unrelated subjects with 22q13 terminal deletion. In seven cases where the availability of DNA and array technology allowed, we mapped deletion boundaries using comparative intensity analysis with single nucleotide polymorphism (SNP) microarrays. Approximate deletion boundaries for three additional cases were derived from clinical or published molecular data. We also examined brain‐imaging studies for a patient with an intragenic SHANK3 mutation. We report the first brain‐imaging data showing that some patients with 22q13 deletions have severe posterior CBVH, and one individual with a SHANK3 mutation has a normal cerebellum. This genotype–phenotype study suggests that the 22q13 deletion phenotype includes abnormal posterior fossa structures that are unlikely to be attributed to SHANK3 disruption. Other genes in the region, including PLXNB2 and MAPK8IP2, display brain expression patterns and mouse mutant phenotypes critical for proper cerebellar development. Future studies of these genes may elucidate their relationship to 22q13.3 deletion phenotypes.

A novel rasopathy caused by recurrent de novo missense mutations in PPP1CB closely resembles Noonan syndrome with loose anagen hair

Noonan syndrome is a rasopathy caused by mutations in multiple genes encoding components of the RAS/MAPK pathway. Despite its variable phenotype, limited genotype–phenotype correlations exist. Noonan syndrome with loose anagen hair (NS‐LAH) is characterized by its distinctive hair anomalies, developmental differences, and structural brain abnormalities and is caused by a single recurrent missense SHOC2 mutation. SHOC2 forms a complex with protein phosphatase 1 (PP1C). Protein phosphatases counterbalance kinases and control activation of signaling proteins, such as the mitogen‐activated protein kinases of the RAS/MAPK pathway. Here we report four patients with de novo missense mutations in protein phosphatase one catalytic subunit beta (PPP1CB), sharing a recognizable phenotype. Three individuals had the recurrent PPP1CB c.146G>C, p.Pro49Arg mutation, the fourth had a c.166G>C, p.Ala56Pro change. All had relative or absolute macrocephaly, low‐set and posteriorly angulated ears, and developmental delay. Slow growing and/or sparse hair and/or an unruly hair texture was present in all. Three individuals had feeding difficulties requiring feeding tubes. One of two males had cryptorchidism, another had pectus excavatum. Short stature was present in three. A female with the recurrent mutation had a Dandy–Walker malformation and optic nerve hypoplasia. Mild ventriculomegaly occurred in all, cerebellar tonsillar ectopia was seen in two and progressed to Chiari 1 malformation in one individual. Based on the combination of phenotypic findings and PPP1CBs effect on RAF dephosphorylation within the RAS/MAPK pathway, this novel condition can be considered a rasopathy, most similar to NS‐LAH. Collectively, these mutations meet the standardized criteria for pathogenicity.

SnapShot: Genetics of Autism

6q23.3 (AHI1*) Jouberin; interacts with b-catenin in cilia Joubert syndrome. (Reduced brain and body size. Cerebellar, retinal, and kidney defects. Most die by P10. Neuron-specific loss leads to depressive-like phenotypes.)7q35-q36.1 (CNTNAP2*)Caspr2, a neurexin family member; clusters voltage-gated K+ channels Recessive EPI syndrome, ASD, ADHD, TS, OCD. (Neuronal migration defects. Reduced GABAergic neurons and decreased cortical synchrony. Seizures. Deficits in social, repetitive behaviors, and USV.) 9q34.13 (TSC1) Hamartin, a growth inhibitory protein that negatively regulates the mTOR pathwayTuberous Sclerosis type I. (Liver and neural tube defects. Die by E12. Abnormal kidney and liver growth in heterozygotes. Variable brain structure, function and behavior abnormalities in conditional mutants. Die at various postnatal ages. Neuron-specific loss causes abnormal spine morphology and cortical excitability. Loss of LTD.)10q23.31 (PTEN) Protein tyrosine phosphatase; negatively regulates the mTOR pathwayCowden disease. (Placenta and germ cell defects. Die by E9.5. Neuron-specific loss alters synaptic physiology. Heterozygotes have prostate, skin and colon defects, and spontaneous tumors. Macrocephaly, neuronal hypertrophy, abnormal social interaction, and increased survival in conditional mutants.)11q13.4 (DHCR7) Final enzyme in cholesterol biosynthetic pathwaySmith-Lemli-Opitz syndrome. (Craniofacial and lung abnormalities. Die by P1. Abnormal cholesterol regulation and enlarged bladders. Hypomorphic mutants are viable and fertile. Compound mutants have fused toes, enlarged ventricles, and 25% embryonic lethality.) 12p13.33 (CACNA1C)a-1 subunit of a voltage-dependent Ca2+ channelTimothy syndrome. (Die embryonically. Impaired pancreatic function. Motor defects and antidepressant-like behavior in heterozygotes; anxiety-like deficits in females. Neuron-specific loss, impaired cognition and LTP.) 15q11.2 (UBE3A) Ubiquitination ligase; targets protein degradation systemAngelman syndrome. (Small brain, seizure susceptibility, motor and learning deficits. Reduced spine density and impaired LTP. Impaired synapse maturation and plasticity.) 16p13.3 (TSC2) Tuberin; which negatively regulates the mTOR pathwayTuberous Sclerosis type II. (Heart, neural tube, and motor defects. Purkinje cell death. Die by E12. Various tumors and axon guidance defects in heterozygotes. Dominant-negative mutant has enhanced anxiety-like behaviors; motor, learning, social behavior deficits.) 17q11.2 (NF1) Neurofibromin; a GTPase activator and negative regulator of RAS signaling Neurofibromatosis. (Macrocephaly, small eyes, and heart defects. Delayed organ development. Embryonic lethal. Increased astrocytes and tumor susceptibility. LTP and learning and memory deficits in heterozygotes.) Xp21.2 (DMD) Dystrophin; cytoskeletal protein bridging ECMDuchenne muscular dystrophy. (Muscle and heart defects in hemizygous males and homozygous females. Reduced fertility. Abnormal retinal electrophysiology and synapse organization, density, and maturation.)Xp21.3 (ARX) Aristaless-related homeobox protein TFLIS, XLID, EPI, ASD. (Hemizygous males die perinatally. Decreased inhibitory synaptic transmission. Males hemizygous for point mutations or triple repeat expansions have seizures. Defects in behavior and GABAergic neuron generation and migration.)Xq27.3 (FMR1) Fragile X mental retardation protein; an RNA-binding protein that traffics mRNA Fragile X syndrome. (Seizures. Enlarged testes in males. Learning and social behavior defects. Dendritic spine abnormalities. Enhanced LTD and impaired LTP. Altered cortical drive and E/I neuronal cortical networks.) Xq28 (MECP2) MeCP2; involved in transcriptional regulation and chromatin organizationRett syndrome. (Brain, breathing, and motor defects in hemizygous males. Mild cognitive and anxiety-like phenotypes in heterozygous females. Various conditional loss and postnatal reduction mimic null phenotypes in adult hemizygous males. Impaired excitatory synapses and spine morphology. Increased neuronal connectivity.)

Zac1 plays a key role in the development of specific neuronal subsets in the mouse cerebellum

BackgroundThe cerebellum is composed of a diverse array of neuronal subtypes. Here we have used a candidate approach to identify Zac1, a tumor suppressor gene encoding a zinc finger transcription factor, as a new player in the transcriptional network required for the development of a specific subset of cerebellar nuclei and a population of Golgi cells in the cerebellar cortex.ResultsWe found that Zac1 has a complex expression profile in the developing cerebellum, including in two proliferating progenitor populations; the cerebellar ventricular zone and the external granular layer overlying posterior cerebellar lobules IX and X. Zac1 is also expressed in some postmitotic cerebellar neurons, including a subset of GABAergic interneurons in the medial cerebellar nuclei. Notably, GABAergic interneurons in the cerebellar nuclei are derived from the cerebellar ventricular zone, where Zac1 is also expressed, consistent with a lineage relationship between these two Zac1+ populations. Zac1 is also expressed in a small subset of cells in the posterior vermis, including some neurogranin-immunoreactive (NG+) Golgi cells, which, based on short-term birthdating, are derived from the EGL, where Zac1 is also expressed. However, Zac1+ cells and NG+ Golgi cells in the cerebellar cortex also display unique properties, as they are generated within different, albeit overlapping, time windows. Finally, consistent with the expression profile of Zac1, two conspicuous abnormalities were found in the cerebellum of Zac1 null mice: the medial cerebellar nuclei, and not the others, were significantly reduced in size; and the number of Golgi cells in cerebellar lobule IX was reduced by approximately 60% compared to wild-type littermates.ConclusionsThe data presented here indicate that the tumor suppressor gene Zac1 is expressed in a complex fashion in the developing cerebellum, including in two dividing progenitor populations and in specific subsets of postmitotic neurons, including Golgi cells and GABAergic neurons in the medial nuclei, which require Zac1 for their differentiation. We thus conclude that Zac1 is a critical regulator of normal cerebellar development, adding a new transcriptional regulator to the growing list of factors involved in generating neuronal diversity in the developing cerebellum.