Susanne Schoch

Molecular organization of the presynaptic active zone

The exocytosis of neurotransmitter-filled synaptic vesicles is under tight temporal and spatial control in presynaptic nerve terminals. The fusion of synaptic vesicles is restricted to a specialized area of the presynaptic plasma membrane: the active zone. The protein network that constitutes the cytomatrix at the active zone (CAZ) is involved in the organization of docking and priming of synaptic vesicles and in mediating use-dependent changes in release during short-term and long-term synaptic plasticity. To date, five protein families whose members are highly enriched at active zones (Munc13s, RIMs, ELKS proteins, Piccolo and Bassoon, and the liprins-α), have been characterized. These multidomain proteins are instrumental for the diverse functions performed by the presynaptic active zone.

Synaptobrevin is essential for fast synaptic-vesicle endocytosis

Synaptobrevin-2 (VAMP-2), the major SNARE protein of synaptic vesicles, is required for fast calcium-triggered synaptic-vesicle exocytosis. Here we show that synaptobrevin-2 is also essential for fast synaptic-vesicle endocytosis. We demonstrate that after depletion of the readily releasable vesicle pool, replenishment of the pool is delayed by knockout of synaptobrevin. This delay was not from a loss of vesicles, because the total number of pre-synaptic vesicles, docked vesicles and actively recycling vesicles was unaffected. However, altered shape and size of the vesicles in synaptobrevin-deficient synapses suggests a defect in endocytosis. Consistent with such a defect, the stimulus-dependent endocytosis of horseradish peroxidase and fluorescent FM1-43 were delayed, indicating that fast vesicle endocytosis may normally be nucleated by a SNARE-dependent coat. Thus, synaptobrevin is essential for two fast synapse-specific membrane trafficking reactions: fast exocytosis for neurotransmitter release and fast endocytosis that mediates rapid reuse of synaptic vesicles.

Transcriptional Upregulation of Cav3.2 Mediates Epileptogenesis in the Pilocarpine Model of Epilepsy

In both humans and animals, an insult to the brain can lead, after a variable latent period, to the appearance of spontaneous epileptic seizures that persist for life. The underlying processes, collectively referred to as epileptogenesis, include multiple structural and functional neuronal alterations. We have identified the T-type Ca2+ channel Cav3.2 as a central player in epileptogenesis. We show that a transient and selective upregulation of Cav3.2 subunits on the mRNA and protein levels after status epilepticus causes an increase in cellular T-type Ca2+ currents and a transitional increase in intrinsic burst firing. These functional changes are absent in mice lacking Cav3.2 subunits. Intriguingly, the development of neuropathological hallmarks of chronic epilepsy, such as subfield-specific neuron loss in the hippocampal formation and mossy fiber sprouting, was virtually completely absent in Cav3.2−/− mice. In addition, the appearance of spontaneous seizures was dramatically reduced in these mice. Together, these data establish transcriptional induction of Cav3.2 as a critical step in epileptogenesis and neuronal vulnerability.

Deletion of CASK in mice is lethal and impairs synaptic function

CASK is an evolutionarily conserved multidomain protein composed of an N-terminal Ca2+/calmodulin-kinase domain, central PDZ and SH3 domains, and a C-terminal guanylate kinase domain. Many potential activities for CASK have been suggested, including functions in scaffolding the synapse, in organizing ion channels, and in regulating neuronal gene transcription. To better define the physiological importance of CASK, we have now analyzed CASK “knockdown” mice in which CASK expression was suppressed by ≈70%, and CASK knockout (KO) mice, in which CASK expression was abolished. CASK knockdown mice are viable but smaller than WT mice, whereas CASK KO mice die at first day after birth. CASK KO mice exhibit no major developmental abnormalities apart from a partially penetrant cleft palate syndrome. In CASK-deficient neurons, the levels of the CASK-interacting proteins Mints, Veli/Mals, and neurexins are decreased, whereas the level of neuroligin 1 (which binds to neurexins that in turn bind to CASK) is increased. Neurons lacking CASK display overall normal electrical properties and form ultrastructurally normal synapses. However, glutamatergic spontaneous synaptic release events are increased, and GABAergic synaptic release events are decreased in CASK-deficient neurons. In contrast to spontaneous neurotransmitter release, evoked release exhibited no major changes. Our data suggest that CASK, the only member of the membrane-associated guanylate kinase protein family that contains a Ca2+/calmodulin-dependent kinase domain, is required for mouse survival and performs a selectively essential function without being in itself required for core activities of neurons, such as membrane excitability, Ca2+-triggered presynaptic release, or postsynaptic receptor functions.

Locomotion, Theta Oscillations, and the Speed-Correlated Firing of Hippocampal Neurons Are Controlled by a Medial Septal Glutamatergic Circuit

Before the onset of locomotion, the hippocampus undergoes a transition into an activity-state specialized for the processing of spatially related input. This brain-state transition is associated with increased firing rates of CA1 pyramidal neurons and the occurrence of theta oscillations, which both correlate with locomotion velocity. However, the neural circuit by which locomotor activity is linked to hippocampal oscillations and neuronal firing rates is unresolved. Here we reveal a septo-hippocampal circuit mediated by glutamatergic (VGluT2(+)) neurons that is activated before locomotion onset and that controls the initiation and velocity of locomotion as well as the entrainment of theta oscillations. Moreover, via septo-hippocampal projections onto alveus/oriens interneurons, this circuit regulates feedforward inhibition of Schaffer collateral and perforant path input to CA1 pyramidal neurons in a locomotion-dependent manner. With higher locomotion speed, the increased activity of medial septal VGluT2 neurons is translated into increased axo-somatic depolarization and higher firing rates of CA1 pyramidal neurons. VIDEO ABSTRACT.

Mutations in POGLUT1, Encoding Protein O-Glucosyltransferase 1, Cause Autosomal-Dominant Dowling-Degos Disease

Dowling-Degos disease (DDD) is an autosomal-dominant genodermatosis characterized by progressive and disfiguring reticulate hyperpigmentation. We previously identified loss-of-function mutations in KRT5 but were only able to detect pathogenic mutations in fewer than half of our subjects. To identify additional causes of DDD, we performed exome sequencing in five unrelated affected individuals without mutations in KRT5. Data analysis identified three heterozygous mutations from these individuals, all within the same gene. These mutations, namely c.11G>A (p.Trp4*), c.652C>T (p.Arg218*), and c.798-2A>C, are within POGLUT1, which encodes protein O-glucosyltransferase 1. Further screening of unexplained cases for POGLUT1 identified six additional mutations, as well as two of the above described mutations. Immunohistochemistry of skin biopsies of affected individuals with POGLUT1 mutations showed significantly weaker POGLUT1 staining in comparison to healthy controls with strong localization of POGLUT1 in the upper parts of the epidermis. Immunoblot analysis revealed that translation of either wild-type (WT) POGLUT1 or of the protein carrying the p.Arg279Trp substitution led to the expected size of about 50 kDa, whereas the c.652C>T (p.Arg218*) mutation led to translation of a truncated protein of about 30 kDa. Immunofluorescence analysis identified a colocalization of the WT protein with the endoplasmic reticulum and a notable aggregating pattern for the truncated protein. Recently, mutations in POFUT1, which encodes protein O-fucosyltransferase 1, were also reported to be responsible for DDD. Interestingly, both POGLUT1 and POFUT1 are essential regulators of Notch activity. Our results furthermore emphasize the important role of the Notch pathway in pigmentation and keratinocyte morphology.

Epilepsy, hippocampal sclerosis and febrile seizures linked by common genetic variation around SCN1A

Epilepsy comprises several syndromes, amongst the most common being mesial temporal lobe epilepsy with hippocampal sclerosis. Seizures in mesial temporal lobe epilepsy with hippocampal sclerosis are typically drug-resistant, and mesial temporal lobe epilepsy with hippocampal sclerosis is frequently associated with important co-morbidities, mandating the search for better understanding and treatment. The cause of mesial temporal lobe epilepsy with hippocampal sclerosis is unknown, but there is an association with childhood febrile seizures. Several rarer epilepsies featuring febrile seizures are caused by mutations in SCN1A, which encodes a brain-expressed sodium channel subunit targeted by many anti-epileptic drugs. We undertook a genome-wide association study in 1018 people with mesial temporal lobe epilepsy with hippocampal sclerosis and 7552 control subjects, with validation in an independent sample set comprising 959 people with mesial temporal lobe epilepsy with hippocampal sclerosis and 3591 control subjects. To dissect out variants related to a history of febrile seizures, we tested cases with mesial temporal lobe epilepsy with hippocampal sclerosis with (overall n = 757) and without (overall n = 803) a history of febrile seizures. Meta-analysis revealed a genome-wide significant association for mesial temporal lobe epilepsy with hippocampal sclerosis with febrile seizures at the sodium channel gene cluster on chromosome 2q24.3 [rs7587026, within an intron of the SCN1A gene, P = 3.36 × 10−9, odds ratio (A) = 1.42, 95% confidence interval: 1.26–1.59]. In a cohort of 172 individuals with febrile seizures, who did not develop epilepsy during prospective follow-up to age 13 years, and 6456 controls, no association was found for rs7587026 and febrile seizures. These findings suggest SCN1A involvement in a common epilepsy syndrome, give new direction to biological understanding of mesial temporal lobe epilepsy with hippocampal sclerosis with febrile seizures, and open avenues for investigation of prognostic factors and possible prevention of epilepsy in some children with febrile seizures.

Differential Pi3K-pathway Activation in Cortical Tubers and Focal Cortical Dysplasias with Balloon Cells

Balloon cells of distinct focal cortical dysplasias type IIb (FCDIIb) and giant cells of cortical tubers in tuberous sclerosis (TSC) constitute neuropathological hallmarks and cytological similarities. In TSC, frequent mutations in the TSC1 or TSC2 genes result in mTOR‐signaling activity. Here, we addressed whether Pi3K‐pathway activation differentiates balloon cells from giant cells. We used immunohistochemistry with antibodies against p‐PDK1 (S241), p‐Akt (S473), p‐tuberin (T1462), p‐p70S6K (T389), p‐p70S6K (T229) and phalloidin‐staining to analyze stress fiber formation in balloon cells of FCDIIb (n = 23) compared with cortical tuber giant cells (n = 5) and adjacent normal CNS tissue as control. We have further established an in vitro assay to assess potential phosphorylation between Akt and S6. We observed phosphorylated (p‐)PDK1, p‐Akt, p‐tuberin, and p‐p70‐kDa S6‐kinase (p‐p70S6K; residue T229) in balloon cells, whereas giant cells showed only equivalent levels of p‐tuberin, p‐p70S6K and stress fibers. Furthermore, Pi3K‐cascade activity in balloon cells may reflect pathway “cross‐talk”. An in vitro assay revealed S6, a major target of p70S6K, to increase phosphorylation of Akt. Our data suggest recruitment of different Pi3K‐cascade factors in the molecular pathogenesis of giant cells in cortical tubers vs. balloon cells in FCDIIb and provides new implications for the development of treatment strategies for these cortical malformations.

Dendritic structural degeneration is functionally linked to cellular hyperexcitability in a mouse model of Alzheimer's disease.

Dendritic structure critically determines the electrical properties of neurons and, thereby, defines the fundamental process of input-to-output conversion. The diversity of dendritic architectures enables neurons to fulfill their specialized circuit functions during cognitive processes. It is known that this dendritic integrity is impaired in patients with Alzheimers disease and in relevant mouse models. It is unknown, however, whether this structural degeneration translates into aberrant neuronal function. Here we use in vivo whole-cell patch-clamp recordings, high-resolution STED imaging, and computational modeling of CA1 pyramidal neurons in a mouse model of Alzheimers disease to show that structural degeneration and neuronal hyperexcitability are crucially linked. Our results demonstrate that a structure-dependent amplification of synaptic input to action potential output conversion might constitute a novel cellular pathomechanism underlying network dysfunction with potential relevance for other neurodegenerative diseases with abnormal changes of dendritic morphology.

Molecular Neuropathology of Temporal Lobe Epilepsy: Complementary Approaches in Animal Models and Human Disease Tissue

Summary:  Patients with temporal lobe epilepsies (TLE) frequently develop pharmacoresistance to antiepileptic treatment. In individuals with drug‐refractory TLE, neurosurgical removal of the epileptogenic focus provides a therapy option with high potential for seizure control. Biopsy specimens from TLE patients constitute unique tissue resources to gain insights in neuropathological and molecular alterations involved in human TLE. Compared to human tissue specimens in most neurological diseases, where only autopsy material is available, the bioptic tissue samples from pharmacoresistant TLE patients open rather exceptional preconditions for molecular biological, electrophysiological as well as biochemical experimental approaches in human brain tissue, which cannot be carried out in postmortem material. Pathological changes in human TLE tissue are multiple and relate to structural and cellular reorganization of the hippocampal formation, selective neurodegeneration, and acquired changes of expression and distribution of neurotransmitter receptors and ion channels, underlying modified neuronal excitability. Nevertheless, human TLE tissue specimens have some limitations. For obvious reasons, human TLE tissue samples are only available from advanced, drug‐resistant stages of the disease. However, in many patients, a transient episode of status epilepticus (SE) or febrile seizures in childhood can induce multiple structural and functional alterations that after a latency period result in a chronic epileptic condition. This latency period, also referred to as epileptogenesis, cannot be studied in human TLE specimens. TLE animal models may be particularly helpful in order to shed characterize new molecular pathomechanisms related to epileptogenesis and open novel therapeutic strategies for TLE. Here, we will discuss experimental approaches to unravel molecular–neuropathological aspects of TLE and highlight characteristics and potential of molecular studies in human and/or experimental TLE.