The role of trained and untrained dogs in the detection and warning of seizures.

Seizure unpredictability plays a major role in disability and decreased quality of life in people with epilepsy. Dogs have been used to assist people with disabilities and have shown promise in detecting seizures. There have been reports of trained seizure-alerting dogs (SADs) successfully detecting when a seizure is occurring or indicating imminent seizures, allowing patients to take preventative measures. Untrained pet dogs have also shown the ability to detect seizures and provide comfort and protection during and after seizures. Dogs' exceptional olfactory abilities and sensitivity to human cues could contribute to their seizure-detection capabilities. This has been supported by studies in which dogs have distinguished between epileptic seizure and non-seizure sweat samples, probably though the detection of volatile organic compounds (VOCs). However, the existing literature has limitations, with a lack of well-controlled, prospective studies and inconsistencies in reported timings of alerting behaviours. More research is needed to standardize reporting and validate the results. Advances in VOC profiling could aid in distinguishing seizure types and developing rapid and unbiased seizure detection methods. In conclusion, using dogs in epilepsy management shows considerable promise, but further research is needed to fully validate their effectiveness and potential as valuable companions for people with epilepsy.

Aromatic l-amino acid decarboxylase deficiency: a patient-derived neuronal model for precision therapies.

Aromatic l-amino acid decarboxylase (AADC) deficiency is a complex inherited neurological disorder of monoamine synthesis which results in dopamine and serotonin deficiency. The majority of affected individuals have variable, though often severe cognitive and motor delay, with a complex movement disorder and high risk of premature mortality. For most, standard pharmacological treatment provides only limited clinical benefit. Promising gene therapy approaches are emerging, though may not be either suitable or easily accessible for all patients. To characterize the underlying disease pathophysiology and guide precision therapies, we generated a patient-derived midbrain dopaminergic neuronal model of AADC deficiency from induced pluripotent stem cells. The neuronal model recapitulates key disease features, including absent AADC enzyme activity and dysregulated dopamine metabolism. We observed developmental defects affecting synaptic maturation and neuronal electrical properties, which were improved by lentiviral gene therapy. Bioinformatic and biochemical analyses on recombinant AADC predicted that the activity of one variant could be improved by l-3,4-dihydroxyphenylalanine (l-DOPA) administration; this hypothesis was corroborated in the patient-derived neuronal model, where l-DOPA treatment leads to amelioration of dopamine metabolites. Our study has shown that patient-derived disease modelling provides further insight into the neurodevelopmental sequelae of AADC deficiency, as well as a robust platform to investigate and develop personalized therapeutic approaches.

LGI1 downregulation increases neuronal circuit excitability.

OBJECTIVE: Leucine-rich glioma-inactivated 1 (LGI1) is a secreted transsynaptic protein that interacts presynaptically with Kv1.1 potassium channels and a disintegrin and metalloprotease (ADAM) protein 23, and postsynaptically influences α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors through a direct link with the ADAM22 cell adhesion protein. Haploinsufficiency of LGI1 or autoantibodies directed against LGI1 are associated with human epilepsy, generating the hypothesis that a subacute reduction of LGI1 is sufficient to increase network excitability. METHODS: We tested this hypothesis in ex vivo hippocampal slices and in neuronal cultures, by subacutely reducing LGI1 expression with shRNA. RESULTS: Injection of shRNA-LGI1 in the hippocampus increased dentate granule cell excitability and low-frequency facilitation of mossy fibers to CA3 pyramidal cell neurotransmission. Application of the Kv1 family blocker, α-dendrotoxin, occluded this effect, implicating the involvement of Kv1.1. This subacute reduction of LGI1 was also sufficient to increase neuronal network activity in neuronal primary culture. SIGNIFICANCE: These results indicate that a subacute reduction in LGI1 potentiates neuronal excitability and short-term synaptic plasticity, and increases neuronal network excitability, opening new avenues for the treatment of limbic encephalitis and temporal lobe epilepsies.

Decanoic acid inhibits mTORC1 activity independent of glucose and insulin signaling.

Low-glucose and -insulin conditions, associated with ketogenic diets, can reduce the activity of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, potentially leading to a range of positive medical and health-related effects. Here, we determined whether mTORC1 signaling is also a target for decanoic acid, a key component of the medium-chain triglyceride (MCT) ketogenic diet. Using a tractable model system, Dictyostelium, we show that decanoic acid can decrease mTORC1 activity, under conditions of constant glucose and in the absence of insulin, measured by phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). We determine that this effect of decanoic acid is dependent on a ubiquitin regulatory X domain-containing protein, mediating inhibition of a conserved Dictyostelium AAA ATPase, p97, a homolog of the human transitional endoplasmic reticulum ATPase (VCP/p97) protein. We then demonstrate that decanoic acid decreases mTORC1 activity in the absence of insulin and under high-glucose conditions in ex vivo rat hippocampus and in tuberous sclerosis complex (TSC) patient-derived astrocytes. Our data therefore indicate that dietary decanoic acid may provide a new therapeutic approach to down-regulate mTORC1 signaling.

Synapsin I Controls Synaptic Maturation of Long-Range Projections in the Lateral Amygdala in a Targeted Selective Fashion.

The amygdala, and more precisely its lateral nucleus, is thought to attribute emotional valence to external stimuli by generating long-term plasticity changes at long-range projections to principal cells. Aversive experience has also been shown to modify pre- and post-synaptic markers in the amygdala, suggesting their possible role in the structural organization of adult amygdala networks. Here, we focused on how the maturation of cortical and thalamic long-range projections occurs on principal neurons and interneurons in the lateral amygdala (LA). We performed dual electrophysiological recordings of identified cells in juvenile and adult GAD67-GFP mice after independent stimulation of cortical and thalamic afferent systems. The results demonstrate that synaptic strengthening occurs during development at synapses projecting to LA principal neurons, but not interneurons. As synaptic strengthening underlies fear conditioning which depends, in turn, on presence and increasing expression of synapsin I, we tested if synapsin I contributes to synaptic strengthening during development. Interestingly, the physiological synaptic strengthening of cortical and thalamic synapses projecting to LA principal neurons was virtually abolished in synapsin I knockout mice, but not differences were observed in the excitatory projections to interneurons. Immunohistochemistry analysis showed that the presence of synapsin I is restricted to excitatory contacts projecting to principal neurons in LA of adult mice. These results indicate that synapsin I is a key regulator of the maturation of synaptic connectivity in this brain region and that is expression is dependent on postsynaptic identity.

Foamy Virus Vectors Transduce Visceral Organs and Hippocampal Structures following In Vivo Delivery to Neonatal Mice.

Viral vectors are rapidly being developed for a range of applications in research and gene therapy. Prototype foamy virus (PFV) vectors have been described for gene therapy, although their use has mainly been restricted to ex vivo stem cell modification. Here we report direct in vivo transgene delivery with PFV vectors carrying reporter gene constructs. In our investigations, systemic PFV vector delivery to neonatal mice gave transgene expression in the heart, xiphisternum, liver, pancreas, and gut, whereas intracranial administration produced brain expression until animals were euthanized 49 days post-transduction. Immunostaining and confocal microscopy analysis of injected brains showed that transgene expression was highly localized to hippocampal architecture despite vector delivery being administered to the lateral ventricle. This was compared with intracranial biodistribution of lentiviral vectors and adeno-associated virus vectors, which gave a broad, non-specific spread through the neonatal mouse brain without regional localization, even when administered at lower copy numbers. Our work demonstrates that PFV can be used for neonatal gene delivery with an intracranial expression profile that localizes to hippocampal neurons, potentially because of the mitotic status of the targeted cells, which could be of use for research applications and gene therapy of neurological disorders.

microRNA targeting of the P2X7 purinoceptor opposes a contralateral epileptogenic focus in the hippocampus.

The ATP-gated ionotropic P2X7 receptor (P2X7R) modulates glial activation, cytokine production and neurotransmitter release following brain injury. Levels of the P2X7R are increased in experimental and human epilepsy but the mechanisms controlling P2X7R expression remain poorly understood. Here we investigated P2X7R responses after focal-onset status epilepticus in mice, comparing changes in the damaged, ipsilateral hippocampus to the spared, contralateral hippocampus. P2X7R-gated inward currents were suppressed in the contralateral hippocampus and P2rx7 mRNA was selectively uploaded into the RNA-induced silencing complex (RISC), suggesting microRNA targeting. Analysis of RISC-loaded microRNAs using a high-throughput platform, as well as functional assays, suggested the P2X7R is a target of microRNA-22. Inhibition of microRNA-22 increased P2X7R expression and cytokine levels in the contralateral hippocampus after status epilepticus and resulted in more frequent spontaneous seizures in mice. The major pro-inflammatory and hyperexcitability effects of microRNA-22 silencing were prevented in P2rx7(-/-) mice or by treatment with a specific P2X7R antagonist. Finally, in vivo injection of microRNA-22 mimics transiently suppressed spontaneous seizures in mice. The present study supports a role for post-transcriptional regulation of the P2X7R and suggests therapeutic targeting of microRNA-22 may prevent inflammation and development of a secondary epileptogenic focus in the brain.

Long-term optical stimulation of channelrhodopsin-expressing neurons to study network plasticity.

Neuronal plasticity produces changes in excitability, synaptic transmission, and network architecture in response to external stimuli. Network adaptation to environmental conditions takes place in time scales ranging from few seconds to days, and modulates the entire network dynamics. To study the network response to defined long-term experimental protocols, we setup a system that combines optical and electrophysiological tools embedded in a cell incubator. Primary hippocampal neurons transduced with lentiviruses expressing channelrhodopsin-2/H134R were subjected to various photostimulation protocols in a time window in the order of days. To monitor the effects of light-induced gating of network activity, stimulated transduced neurons were simultaneously recorded using multi-electrode arrays (MEAs). The developed experimental model allows discerning short-term, long-lasting, and adaptive plasticity responses of the same neuronal network to distinct stimulation frequencies applied over different temporal windows.