Ste20-like kinase is critical for inhibitory synapse maintenance and its deficiency confers a developmental dendritopathy

SummaryThe size and structure of the dendritic arbor play important roles in determining how synaptic inputs of neurons are converted to action potential output. The regulatory mechanisms governing the development of dendrites, however, are insufficiently understood. The evolutionary conserved Ste20/Hippo kinase pathway has been proposed to play an important role in regulating the formation and maintenance of dendritic architecture. A key element of this pathway, Ste20-like kinase (SLK), regulates cytoskeletal dynamics in non-neuronal cells and is strongly expressed throughout neuronal development. However, its function in neurons is unknown. We show that during development of mouse cortical neurons, SLK has a surprisingly specific role for proper elaboration of higher, ≥ 3rd, order dendrites. Moreover, we demonstrate that SLK is required to maintain excitation-inhibition balance. Specifically, SLK knockdown caused a selective loss of inhibitory synapses and functional inhibition after postnatal day 15, while excitatory neurotransmission was unaffected. Finally, we show that this mechanism may be relevant for human disease, as dysmorphic neurons within human cortical malformations revealed significant loss of SLK expression. Overall, the present data identify SLK as a key regulator of both dendritic complexity during development and of inhibitory synapse maintenance.

Dendritic pathology, spine loss and synaptic reorganization in human cortex from epilepsy patients

Neuronal dendritic arborizations and dendritic spines are crucial for a normal synaptic transmission and may be critically involved in the pathophysiology of epilepsy. Alterations in dendritic morphology and spine loss mainly in hippocampal neurons have been reported both in epilepsy animal models and in human brain tissues from patients with epilepsy. However, it is still unclear whether these dendritic abnormalities relate to the cause of epilepsy or are generated by seizure recurrence. We investigated fine neuronal structures at the level of dendritic and spine organization using Golgi impregnation, and analysed synaptic networks with immunohistochemical markers of glutamatergic (vGLUT1) and GABAergic (vGAT) axon terminals in human cerebral cortices derived from epilepsy surgery. Specimens were obtained from 28 patients with different neuropathologically defined aetiologies: type Ia and type II focal cortical dysplasia, cryptogenic (no lesion) and temporal lobe epilepsy with hippocampal sclerosis. Autoptic tissues were used for comparison. Three-dimensional reconstructions of Golgi-impregnated neurons revealed severe dendritic reshaping and spine alteration in the core of the type II focal cortical dysplasia. Dysmorphic neurons showed increased dendritic complexity, reduction of dendritic spines and occasional filopodia-like protrusions emerging from the soma. Surprisingly, the intermingled normal-looking pyramidal neurons also showed severe spine loss and simplified dendritic arborization. No changes were observed outside the dysplasia (perilesional tissue) or in neocortical postsurgical tissue obtained in the other patient groups. Immunoreactivities of vGLUT1 and vGAT showed synaptic reorganization in the core of type II dysplasia characterized by the presence of abnormal perisomatic baskets around dysmorphic neurons, in particular those with filopodia-like protrusions, and changes in vGLUT1/vGAT expression. Ultrastructural data in type II dysplasia highlighted the presence of altered neuropil engulfed by glial processes. Our data indicate that the fine morphological aspect of neurons and dendritic spines are normal in epileptogenic neocortex, with the exception of type II dysplastic lesions. The findings suggest that the mechanisms leading to this severe form of cortical malformation interfere with the normal dendritic arborization and synaptic network organization. The data argue against the concept that long-lasting epilepsy and seizure recurrence per se unavoidably produce a dendritic pathology.

Genetic characterization identifies bottom-of-sulcus dysplasia as an mTORopathy

ObjectiveTo determine the genetic basis of bottom-of-sulcus dysplasia (BOSD), which is a highly focal and epileptogenic cortical malformation in which the imaging, electrophysiologic, and pathologic abnormalities are maximal at the bottom of sulcus, tapering to a normal gyral crown.MethodsTargeted panel deep sequencing (>500×) was performed on paired blood and brain-derived genomic DNA from 20 operated patients with drug-resistant focal epilepsy and BOSD. Histopathology was assessed using immunohistochemistry.ResultsBrain-specific pathogenic somatic variants were found in 6 patients and heterozygous pathogenic germline variants were found in 2. Somatic variants were identified in MTOR and germline variants were identified in DEPDC5 and NPRL3. Two patients with somatic MTOR variants showed a mutation gradient, with higher mutation load at the bottom of sulcus compared to the gyral crown. Immunohistochemistry revealed an abundance of dysmorphic neurons and balloon cells in the bottom of sulcus but not in the gyral crown or adjacent gyri.ConclusionsBOSD is associated with mTOR pathway dysregulation and shares common genetic etiologies and pathogenic mechanisms with other forms of focal and hemispheric cortical dysplasia, suggesting these disorders are on a genetic continuum.

Same same but different: a web-based deep learning application for the histopathologic distinction of cortical malformations

We trained a convolutional neural network (CNN) to classify H.E. stained microscopic images of focal cortical dysplasia type IIb (FCD IIb) and cortical tuber of tuberous sclerosis complex (TSC). Both entities are distinct subtypes of human malformations of cortical development that share histopathological features consisting of neuronal dyslamination with dysmorphic neurons and balloon cells. The microscopic review of routine stainings of such surgical specimens remains challenging. A digital processing pipeline was developed for a series of 56 FCD IIb and TSC cases to obtain 4000 regions of interest and 200.000 sub-samples with different zoom and rotation angles to train a CNN. Our best performing network achieved 91% accuracy and 0.88 AUCROC (area under the receiver operating characteristic curve) on a hold-out test-set. Guided gradient-weighted class activation maps visualized morphological features used by the CNN to distinguish both entities. We then developed a web application, which combined the visualization of whole slide images (WSI) with the possibility for classification between FCD IIb and TSC on demand by our pretrained and build-in CNN classifier. This approach might help to introduce deep learning applications for the histopathologic diagnosis of rare and difficult-to-classify brain lesions.

Dysmorphic neuron density underlies intrinsic epileptogenicity of the centre of cortical tubers

Cortical tubers are benign lesions that develop in patients with tuberous sclerosis complex (TSC), often resulting in drug-resistant epilepsy. Surgical resection may be required for seizure control, but the extent of the resection required is unclear. Many centres include resection of perituberal cortex, which may be associated with neurological deficits. Also, patients with tubers in eloquent cortex may be excluded from epilepsy surgery.Our electrophysiological and MRI studies indicate that the tuber centre is the source of seizures, suggesting that smaller resections may be sufficient for seizure control. Here we report five epilepsy surgeries in four children with TSC and focal motor seizures from solitary epileptogenic tubers in the sensorimotor cortex in whom the resection was limited to the tuber centre, leaving the tuber rim and surrounding perituberal cortex intact. Seizures were eliminated in all cases, and no functional deficits were observed. On routine histopathology we observed an apparent increase in density of dysmorphic neurons at the tuber centre, which we confirmed using unbiased stereology which demonstrated a significantly greater density of dysmorphic neurons within the resected tuber centre (1951 ± 215 cells/mm3) compared to the biopsied tuber rim (531 ± 189 cells/mm3, n = 4, p = 0.008).Taken together with our previous electrophysiological and MRI studies implicating the tuber centre as the focus of epileptic activity, and other electrophysiological studies of dysmorphic neurons in focal cortical dysplasia, this study supports the hypothesis that dysmorphic neurons concentrated at the tuber centre are the seizure generators in TSC. Furthermore, our results support limiting resection to the tuber centre, decreasing the risk of neurological deficits when tubers are located within eloquent cortex.

Novel pathological abnormalities of deep brain structures including dysplastic neurons in anterior striatum associated with focal cortical dysplasia in epilepsy

Object Some patients are not seizure free even after epileptogenic cortical resection. The authors recently described a case of frontal lobe epilepsy cured after the resection of periventricular white matter and striatum, in which dysplastic neurons were revealed. The authors attempted to confirm similar cases. Methods They reviewed the records of 8 children with frontal lobe epilepsy who had daily (7) or monthly (1) seizures and underwent resections including deep brain structures. Results Five patients underwent multiple resections. Neuroimaging of the deep structures showed the transmantle sign in 3 patients, ictal hyperperfusion in 6, reduced iomazenil uptake in 2, and spike dipole clustering in 6. All patients became seizure free postoperatively. Focal cortical dysplasia of various types was diagnosed in all patients. Dysmorphic neurons were found in the cortex and subcortical white matter of 5 patients. The striatum was verified in 3 patients in whom dysmorphic neurons were scattered. In the periventricular white matter, prominent astrocytosis was evident in all cases. Conclusions Pathological abnormalities such as dysmorphic neurons and astrocytosis in deep brain structures would play a key role in epileptogenesis.