The neuropathology of autism: defects of neurogenesis and neuronal migration, and dysplastic changes

Increased brain mass in autistic children and some autistic adults [89], increase in the numerical density of neurons [13, 14], reduced size of neurons [7] and brain structure-specific delay of neuronal growth [111] indicate alterations in neuronal and brain growth in autistic individuals. The subventricular zone of the lateral ventricles [26] and the dentate gyrus [33] are active sites of neurogenesis in adult humans. Several of our findings support the hypothesis of altered neurogenesis in autistic subjects. The increased thickness of the subependymal cell layer, subependymal nodular dysplasia, abnormal growth of the dentate nucleus and dysplasia of the granule layer in the dentate gyrus, detected in this study, appear to be signs of abnormal neurogenesis in the brains of three autistic subjects. Subependymal nodules were reported in approximately 80% of patients with tuberous sclerosis, a disorder that is highly associated with epilepsy, autism and mental retardation [73]. Tuberous sclerosis nodules were detected in one fetus [12], suggesting that fetal development of subependymal nodules can lead to the early onset of epilepsy that was diagnosed at the age of 14 months in a neuropathologically examined autistic male. The subependymal nodules detected in this autistic male’s brain are partially similar to tubers seen in subjects diagnosed with tuberous sclerosis [24]. The cause of subependymal nodular dysplasia in the examined subject is unknown. In the reported subjects, bilateral periventricular nodules are linked to mutations of the filamin A (FLNA) gene located on chromosome Xp28. Filamin A is an actin-crosslinking protein that is essential for cell locomotion [16], and nodule formation might be related to a defect in cell migration. The presence of miniature nodules that were built of poorly differentiated small neurons within the subependymal cell layer and an increase in nodular size with signs of growth and differentiation of neurons suggests that neurogenesis, differentiation and maturation of neurons were in progress within the subependymal germinal matrix of the 7-year-old autistic child. This interpretation of subependymal nodule genesis is consistent with lineage studies demonstrating that cells in nodules express cellular markers that are typical for progenitors derived from the subventricular germinal zone [35, 67]. However, in contrast to the subependymal nodules seen in subjects with tuberous sclerosis, in the examined autistic subject, the nodules seen were small (from 258 to 3,310 μm in diameter), and did not have the characteristic ovoid or polygonal giant cells, 80–150 μm in diameter, giant cells with multiple and peripherally displaced nuclei [25], or balloon cells, which are considered the sine qua non histopathological features of the cortical tubers and subependymal nodules observed in tuberous sclerosis [73].

The enlarged caudate nucleus detected in the brain of the 7-year-old autistic subject is consistent with MRI reports documenting an increased volume of basal ganglia, including the caudate, in autism [54, 55, 66, 99]. A disproportionate increase of the caudate nucleus volume [66] suggests that in brains of some autistic individuals, extended neurogenesis within the subependymal cell layer may contribute to abnormal growth of the caudate nucleus. A similar process has been observed in the brains of people with Huntington disease, showing enhanced neurogenesis in the subependymal layer and suggesting renewal of the neuronal population in a degenerating caudate nucleus [26]. The caudate nucleus is a part of the fronto-striatal network involved in several functional domains that are impaired in autism, including lower order repetitive motor behavior; intense circumscribed patterns of interests and higher order rituals and compulsions [41], and defects in cognitive functions [19, 109], planning and problem-solving skills [78, 98], short- and long-term memory [40] and learning [88].

Heterotopia is a sign of altered migration leading to an abnormal distribution of gray matter nodular masses with disorganized or rudimentary lamination within the periventricular area (periventricular heterotopia) or subcortical white matter (subcortical heterotopia) [2]. In the examined cohorts, heterotopias were detected in the brains of four autistic subjects and in the brain of one control subject. Heterotopias are associated with mutations in the filamin 1 gene (FLNA1) [39, 46] and the chromosome X-linked DCX gene that codes for doublecortin, a protein expressed during brain development in migrating neurons, and in the cortical plate [29, 44, 45], which is involved in the formation of the microtubules necessary for neuronal migration [15]. Periventricular nodular heterotopia has been reported to be associated with pharmaco-resistant seizures in 80–90% of patients [31]. In the examined cohort, two periventricular heterotopias were detected in the brain of a child with subependymal nodular dysplasia and seizures diagnosed at 14 months of age (B-6403). Early onset epilepsy, diagnosed at the age of 4.5 months, might be related to the multiple heterotopias found within the frontal inferior gyrus, vermis and cerebellar white matter, coexisting with a focal cortical dysplasia and dentate gyrus dysplasia (B-5342).

The most common form of developmental changes detected in the examined brains was focal dysplasia, which was observed in 11 (85%) of the autistic subjects. The morphology of focal dysplasias appears to reflect signs of abnormal migration, neuronal immaturity and altered cell arrangement, resulting in focal distortion of cytoarchitecture. In spite of similarities, the dysplastic changes in the neocortex and archicortex, dentate gyrus and cornu Ammonis and cerebellum also reveal a brain structure-specific pattern of dysplastic changes in autism.

Dysplasias encompass a spectrum of changes ranging from a mild form of cortical disruption, without cellular abnormalities, to the most severe form with cortical dyslamination, with abnormal morphology of neurons and astrocytes [93, 96, 107]. Focal cortical dysplasias with giant neurons and balloon cells [107, 113] are histopathologically similar to tubers containing giant cells in tuberous sclerosis complex [25, 73], suggesting a common pathogenic basis [113]. However, activation of the mammalian target of rapamycin (mTOR) pathway observed in the tuberous sclerosis complex is not present in focal cortical dysplasia [8, 80]. The giant neurons and ballooned cells, which are histopathological features of tuberous sclerosis and focal cortical dysplasia, were absent both in the subependymal nodules and in the focal cortical dysplasia observed in the examined autistic cohort. These findings suggest that in spite of similarities, the pathomechanisms of developmental alterations are different in the examined autistic subjects than those in tuberous sclerosis heterotopias or focal cortical dysplasia. The development of the giant neuron- and balloon cell-free dysplasias observed in the autistic subjects might be related to differences in cause and/or mechanism. The detection of changes similar to focal cortical dysplasia in association with prenatal ischemia [65] or in shaken infant syndrome [74] may support these speculations.

Ectopias and dysplastic changes were reported in the brains of autistic subjects, by several groups [4, 62‐64, 91]. Bailey et al. [4] detected olivary dysplasia in the brain of three of the five autistic subjects, and ectopic neurons related to the olivary complex in two cases. Moreover, in the brains of four autistic subjects, cortical dysgenesis was found. In the brains of the autistic subjects, a strikingly consistent finding was cingulate cortex disordered lamination [62‐64, 100]. A recent study of the cingulate cortex of nine autistic subjects revealed a developmental malformation with irregular lamination in three cases, and an increased number of neurons within the subcortical white matter in two [100]. Simms et al. [100] suggest that the excessive number of neurons in the subcortical white matter reflects the lack of proper resolution of the transient zone in the developing brain of autistic subjects. Studies by Fatemi et al. [37, 38] link the migration and lamination defects to a striking reduction of reelin (by 40%) and Bcl-2 (by 34–51%) in the brains of autistic subjects. Our studies along with others’ suggest that in the majority of autistic subjects, heterotopias and dysplastic changes are the local sign of general developmental defects of migration with a broad spectrum of topographic, morphological, and functional outcomes.

In the examined brains of autistic subjects, signs of neuronal immaturity were a common finding. Failure of maturation of neuronal precursors caused by altered expression of cytoskeletal proteins and loss of neuronal polarity results in defects in migration to the destined layer and in incorrect vertical and horizontal orientation [93]. The immaturity of dysplastic neurons is reflected in the expression of a variety of proteins and mRNA that are not present in mature neurons an altered expression of developmentally regulated cytoskeletal elements [3, 23, 61, 76], which are known to be crucial for dendrite arborization, spine formation, axon outgrowth and maintenance of cell size and shape. Reduced cell size, dendritic arborization and spine expression are characteristic of dysplastic neurons [6, 93]. Cortical dysplasias are the most epileptogenic lesions of the brain [107] and are observed in up to 25% of all epileptic surgeries [102]. More subtle cortical malformations or dysgenesis encountered in adults with epilepsy may lack the histological criteria for focal cortical dysplasia. They have been described as mild cortical dysplasia or microdysgenesis [77].

Microdysgenesis within the entorhinal cortex of the 23- and the 60-year-old autistic subjects in the examined cohort is unique because the selective deficit of neurons was limited almost exclusively to the stellate neurons in the second layer. It is possible that the observed dysgenesis is a result of defective migration of neurons to their intended destinations. The presence of a thicker molecular layer and the deeper location of islands in the entorhinal cortex of subjects with schizophrenia were previously interpreted as evidence that the stellate neurons do not reach their destinations during development, probably due to abnormal migration [36, 57]. Studies indicating the involvement of reelin and Bcl2 genes in the pathogenesis of schizophrenia [37, 47, 60] and the reduced expression of reelin and Bcl2 in people with autism suggest that these two genes play a role in abnormal brain development and contribute to the structural and functional anomalies seen in autism and schizophrenia [37].

The distortion of dentate gyrus development detected in two autistic subjects was reflected in granule cell migration into the molecular layer and formation of an additional granule cell layer. Distortion of the shape of the dentate granule cell layer with the formation of irregular circles and loops appears to be another piece of evidence suggesting abnormal neuronal migration and networking. Numerous factors up-regulate neurogenesis in the hippocampus [32], including seizures [70, 71], antidepressant drugs [59, 72] and lithium [18]. Several areas of dysplastic changes with significant deficits of pyramidal neurons were found in the CA1 sector in three autistic subjects, but thickening of the pyramidal layer and an increased packing of dysplastic neurons in the CA1 sector of the 56-year-old subject suggests a diversity of CA dysplasia patterns in autism. The lack of gliosis indicates that the observed pathology is a sign of microdysgenesis rather than an effect of hypoxic neuronal loss. A significant deficit of mature pyramidal neurons and the presence of small irregular or poorly differentiated oval neurons suggest the defect of neuronal maturation in autism.

We report a spectrum of focal developmental changes seen in the cerebellum of eight autistic subjects, including nodular (lobe X) [97] dysplasia in the cerebellum in five, vermal dysplasia in one, severe focal hypoplasia in one, and heterotopias in one other subject. The presence of heterotopias only in one control subject is evidence of a strong tendency for focal developmental changes of cerebellar microarchitecture that were present in 61% of the autistic subjects. Flocculonodular dysplasia affecting almost the entire lobe indicates that mechanisms leading to focal dysplasia, which were present in five (38%) of the autistic subjects, show extremely strong topographic predilection. The observed focal dysplasia was associated with profound local disorganization of granule cells, Purkinje cells and molecular layers limited to a small cerebellar compartment receiving major projections from the vestibular complex involved in the oculomotor and postural system. Similar cerebellar dysplastic changes classified as heterotaxias (clusters of poorly organized mixed cells) were identified in 14% of normal infants but in 83% of infants with trisomy of different chromosomes [92]. The presence within the dysplastic nodule of both GABAergic Purkinje cells produced from the cerebellar ventricular zone, and the glutamatergic granule neurons produced from the rhombic lip, and the preservation of the cytoarchitecture in the adjacent cerebellar folia suggest that the final steps of migration and networking are disturbed mainly or exclusively in the nodule of the majority of autistic subjects. The characteristic feature distinguishing lobule X from the other lobules is the abundance of the transcription factor Tbr2 positive unipolar brush cells (UBCs) [30, 34], which amplify inputs from vestibular ganglia and nuclei, by spreading and prolonging excitation within the internal granular layer [84]. Abnormal networking of Purkinje cells, granule neurons, and UBCs may contribute to altered cerebellar coordination of locomotion and motor learning and planning, as well as of higher cognitive processing [58]. Flocculonodular dysplasia appears to be another sign of the mosaic of local developmental defects, most likely predetermined by the spatial patterning of germinal zones in developing rhombic lip [110], and coexisting with more general developmental defects resulting in the accelerated growth of the brain in early childhood [89], minicolumn pathology [13, 14], reduced neuron volume [7, 108, 111], and desynchronized neuronal growth in many brain regions [111] observed in autism.

Identification of sub-groups with signs of hyperplasia, hypoplasia and normal-sized cerebellum [95] reflects the heterogeneity of the autistic population. Piven et al. [87] reported that cerebellar volume correlates with an increased total brain volume. In the majority of autistic subjects, reduced size of the cerebellar hemisphere is observed [42, 82], but this trend is not detectable in cohorts of high-functioning autistic individuals [56]. Regional hypoplasia affects the vermis in autistic individuals relatively often [20, 22, 52] and may be associated with the deficits in attention-orienting [49, 104], stereotypic behavior and reduced exploration observed in autism [86]. In the examined autistic cohort, selective and severe hypoplasia of lobes 1–4 associated with hypoconvolution of a large portion of the dentate nucleus appears to correspond to clinically detected defects of movement coordination. These findings suggest that differences in the type, topography and severity of cerebellar developmental defects may contribute to different clinical manifestations.

In the 4–7-year-old autistic children examined in this study, the volume of the Purkinje cells was 38% smaller than that of the age-matched control group [111]. Moreover, it has been reported that Purkinje cells of the autistic subjects revealed a 40% decrease in the expression of glutamic acid decarboxylase 67 (GAD67) mRNA [114]. In autism, the basket cells provide an increased GABAergic feed-forward inhibition to Purkinje cells. The result could be disruption in the timing of Purkinje cell firings and altered inhibition of the cerebellar nuclei, which could directly affect cerebello-cortical output and contribute to the changes in motor behavior and cognition observed in autism [115]. These findings and the reduced volume (by 26%) of the neurons of the dentate nucleus seen in the 4–7-year-old autistic children [111] suggest that in autism, interactions between the Purkinje cells and dentate nucleus are modified on the structural, molecular and functional levels.

The (a) detected changes within the subependymal cell layer with subependymal nodular dysplasia, (b) subcortical and periventricular heterotopias and (c) neocortex, archicortex, dentate gyrus, cornu Ammonis and cerebellar dysplasia reflect focal modification of neurogenesis, migration and alterations of the cytoarchitecture of brain cortex, subcortical structures and cerebellum in autism. Detection of dysplastic changes only in one control brain and of the broad spectrum of focal developmental alterations in the brains of 92% of the autistic subjects indicates that focal changes are a reflection of global developmental abnormalities and that regional changes may have their own contribution to the clinical heterogeneity of autism.