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Neuropathological evaluation and granule cell layer with biocytin-filled cell: Example of (a–c) H&E and (d–f) MAP2 staining in hippocampal tissue resected from a patient with TLE. The sectioning and staining are made for neuropathological evaluation showing (a,d) the whole slice including dentate gyrus, CA3-CA1 and subiculum. The electrophysiological experiments were performed in the dentate gyrus magnified in B and E with the granule cell layer (gcl) identified, and further magnification inset showing individual granule cells. (c,f) In CA1, the neuronal cell loss is clear, the pyramidal cell layer has completely disappeared. After electrophysiological experiments the slices were fixated in PFA and later stained for (g) MAP2 (red) showing the granule cell layer with the recorded biocytin-filled granule cell (in green). (h) Higher magnification of the recorded neuron with typical granule cell morphology. Scale bar: 2 mm in a and d, 150 µm in b, c, e and f with 50 µm in inset, 200 µm in g and 50 µm in h.
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In epilepsy patients, drug-resistant seizures often originate in one of the temporal lobes. In selected cases, when certain requirements are met, this area is surgically resected for therapeutic reasons. We kept the resected tissue slices alive in vitro for 48 h to create a platform for testing a novel treatment strategy based on neuropeptide Y (NP...
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Loss of neuropeptide Y (NPY)-expressing interneurons in the hippocampus and decaying cholinergic neuromodulation are thought to contribute to impaired cognitive function during aging. However, the interaction of these two neuromodulatory systems in maintaining hippocampal synaptic plasticity during healthy aging has not been explored so far. Here w...
Citations
... Pam has previously been found to be expressed in astrocytes 31,32 and is transiently increased in the hippocampus in kainic acid (KA) seizures in rats 33 , however, it has not been identified in TBI epileptogenesis. Interestingly, it is involved in the production of amidated neuropeptide Y, which has been shown to exert a significant inhibitory effect on epileptiform activity in the human hippocampal dentate gyrus 34 . It is plausible that increased Pam expression in astrocytes may be the result of a demand for amidated peptides, needed to regulate astrocytic functions such as proliferation 35 or to counteract seizure development. ...
Post-traumatic epilepsy (PTE) stands as one of the numerous debilitating consequences that follow traumatic brain injury (TBI). Despite its impact on many individuals, the current landscape offers only a limited array of reliable treatment options, and our understanding of the underlying mechanisms and susceptibility factors remains incomplete. Among the potential contributors to epileptogenesis, astrocytes, a type of glial cell, have garnered substantial attention as they are believed to promote hyperexcitability and the development of seizures in the brain following TBI. The current study evaluated the transcriptomic changes in cortical astrocytes derived from animals that developed seizures as a result of severe focal TBI. Using RNA-Seq and ingenuity pathway analysis (IPA), we unveil a distinct gene expression profile in astrocytes, including alterations in genes supporting inflammation, early response modifiers, and neuropeptide-amidating enzymes. The findings underscore the complex molecular dynamics in astrocytes during PTE development, offering insights into therapeutic targets and avenues for further exploration.
... This effect is consistent with previous electrophysiological studies that have shown exogenous application of NPY can suppress pathogenic excitability in epilepsy models. 63,64 Exvivo we confirm that NPY can impact the excitability of not only excitatory neurons, presumed corticomotoneurons, within layer 5 of the motor cortex, but also the inhibitory neurons within this main output layer of the motor cortex. After bath application of NPY, interneurons had a lower rheobase and could fire more readily in response to a given current, while in contrast, excitatory neurons fired less readily, with an increased rheobase. ...
Objective:
Neuropeptide Y (NPY) is a 36 amino acid peptide widely considered to provide neuroprotection in a range of neurodegenerative diseases. In the fatal motor neuron disease amyotrophic lateral sclerosis (ALS), recent evidence supports a link between NPY and ALS disease processes. The goal of this study was to determine the therapeutic potential and role of NPY in ALS, harnessing the brain-targeted intranasal delivery of the peptide, previously utilised to correct motor and cognitive phenotypes in other neurological conditions.
Methods:
To confirm the association with clinical disease characteristics, NPY expression was quantified in post-mortem motor cortex tissue of ALS patients and age-matched controls. The effect of NPY on ALS cortical pathophysiology was investigated using slice electrophysiology and multi-electrode array recordings of SOD1G93A cortical cultures in vitro. The impact of NPY on ALS disease trajectory was investigated by treating SOD1G93A mice intranasally with NPY and selective NPY receptor agonists and antagonists from pre-symptomatic and symptomatic phases of disease.
Results:
In the human post-mortem ALS motor cortex, we observe a significant increase in NPY expression, which is not present in the somatosensory cortex. In vitro, we demonstrate that NPY can ameliorate ALS hyperexcitability, while brain-targeted nasal delivery of NPY and a selective NPY Y1 receptor antagonist modified survival and motor deficits specifically within the symptomatic phase of the disease in the ALS SOD1G93A mouse.
Interpretation:
Taken together, these findings highlight the capacity for non-invasive brain-targeted interventions in ALS and support antagonism of NPY Y1Rs as a novel strategy to improve ALS motor function.
... Human temporal lobe (hippocampus) tissue was obtained by surgical resections from three patients treated for intractable epilepsy at Rigshospitalet in Copenhagen, Denmark, as previously described [38,39]. Prior to each surgery a written informed consent was obtained from all subjects. ...
... We have previously demonstrated that in hippocampal slices surgically removed from patients with drug-resistant TLE, NPY application decreased excitatory synaptic transmission and epileptiform activity, supporting an antiepileptic effect of NPY in human epileptic tissue [38,39]. To further investigate translational validity of the AAV1-NPY/Y2 vector, it seemed important to demonstrate that the vector was capable of expressing the transgenes in the human tissue. ...
Gene therapy with AAV vectors carrying genes for neuropeptide Y and its receptor Y2 has been shown to inhibit seizures in multiple animal models of epilepsy. It is however unknown how the AAV serotype or the sequence order of these two transgenes in the expression cassette affects the actual parenchymal gene expression levels and the seizure-suppressant efficacy. To address these questions, we compared three viral vector serotypes (AAV1, AAV2 and AAV8) and two transgene sequence orders (NPY-IRES-Y2 and Y2-IRES-NPY) in a rat model of acutely induced seizures. Wistar male rats were injected bilaterally with viral vectors and 3 weeks later acute seizures were induced by a subcutaneous injection of kainate. The latency until 1st motor seizure, time spent in motor seizure and latency to status epilepticus were measured to evaluate the seizure-suppressing efficacy of these vectors compared to an empty cassette control vector. Based on the results, the effect of the AAV1-NPY-IRES-Y2 vector was further investigated by in vitro electrophysiology, and its ability to achieve transgene overexpression in resected human hippocampal tissue was evaluated. The AAV1-NPY-IRES-Y2 proved to be better to any other serotype or gene sequence considering both transgene expression and ability to suppress induced seizures in rats. The vector also demonstrated transgene-induced decrease of glutamate release from excitatory neuron terminals and significantly increased both NPY and Y2 expression in resected human hippocampal tissue from patients with drug-resistant temporal lobe epilepsy. These results validate the feasibility of NPY/Y2 receptor gene therapy as a therapeutic opportunity in focal epilepsies.
... Thus, the lack of Zn 2+ inhibition in the hippocampus can trigger a cascade of neuronal hyperexcitability leading to epileptiform activity and seizures. NPY suppresses epileptiform activity and its expression is upregulated in response to seizures (78,79). Consequently, our observation of age-dependent increased NPY expression in the CA3 is accordant with the removal of Zn 2+ enhancement of inhibitory response, as is our observation of age-dependent decrease of calbindin, a cytoplasmic protein that buffers intraneuronal calcium and is downregulated by seizures in the DG region but not in CA1 (80). ...
Vesicular Zn²⁺ (zinc) is released at synapses and has been demonstrated to modulate neuronal responses. However, mechanisms through which dysregulation of zinc homeostasis may potentiate neuronal dysfunction and neurodegeneration are not well-understood. We previously reported that accumulation of soluble amyloid beta oligomers (AβO) at synapses correlates with synaptic loss and that AβO localization at synapses is regulated by synaptic activity and enhanced by the release of vesicular Zn²⁺ in the hippocampus, a brain region that deteriorates early in Alzheimer's disease (AD). Significantly, drugs regulating zinc homeostasis inhibit AβO accumulation and improve cognition in mouse models of AD. We used both sexes of a transgenic mouse model lacking synaptic Zn²⁺ (ZnT3KO) that develops AD-like cognitive impairment and neurodegeneration to study the effects of disruption of Zn²⁺ modulation of neurotransmission in cognition, protein expression and activation, and neuronal excitability. Here we report that the genetic removal of synaptic Zn²⁺ results in progressive impairment of hippocampal-dependent memory, reduces activity-dependent increase in Erk phosphorylation and BDNF mRNA, alters regulation of Erk activation by NMDAR subunits, increases neuronal spiking, and induces biochemical and morphological alterations consistent with increasing epileptiform activity and neurodegeneration as ZnT3KO mice age. Our study shows that disruption of synaptic Zn²⁺ triggers neurodegenerative processes and is a potential pathway through which AβO trigger altered expression of neurotrophic proteins, along with reduced hippocampal synaptic density and degenerating neurons, neuronal spiking activity, and cognitive impairment and supports efforts to develop therapeutics to preserve synaptic zinc homeostasis in the brain as potential treatments for AD.
... For the human tissue experiment, epileptic human hippocampal slices were cut and maintained from tissue received from surgical resections performed at Lund University Hospital and Rigshospitalet University Hospital, as previously described [42,43]. Briefly, resected tissue was collected in an ice-cold sucrose-based cutting solution containing (in mM): 200 sucrose, 21 NaHCO3, 10 glucose, 3 KCl, 1.25 NaH2PO4, 1.6 CaCl2, 2 MgCl2, 2 MgSO4 (all from Sigma-Aldrich), adjusted to 300-310 mOsm, 7.4 pH. ...
Glial cell line-derived neurotrophic factor (GDNF) has been shown to counteract seizures when overexpressed or delivered into the brain in various animal models of epileptogenesis or chronic epilepsy. The mechanisms underlying this effect have not been investigated. We here demonstrate for the first time that GDNF enhances GABAergic inhibitory drive onto mouse pyramidal neurons by modulating postsynaptic GABAA receptors, particularly in perisomatic inhibitory synapses, by GFRα1 mediated activation of the Ret receptor pathway. Other GDNF receptors, such as NCAM or Syndecan3, are not contributing to this effect. We observed similar alterations by GDNF in human hippocampal slices resected from epilepsy patients. These data indicate that GDNF may exert its seizure-suppressant action by enhancing GABAergic inhibitory transmission in the hippocampal network, thus counteracting the increased excitability of the epileptic brain. This new knowledge can contribute to the development of novel, more precise treatment strategies based on a GDNF gene therapy approach.
... It regulates homeostatic processes such as mood, appetite, angiogenesis, cerebral vasospasm, hypothalamic-pituitary axis, and sympathetic and parasympathetic activation. NPY functions as an antiepileptic chemical messenger that inhibits neuronal excitability and seizures [42]. The loss of NPY-positive cells in the hippocampus increases the severity of seizure activity in animals with posttraumatic epilepsy. ...
... Neuropeptide Y (NPY) inhibits seizure-like activity on resected hippocampal tissues in patients treated for drug-resistant temporal lobe epilepsy. Initially, the resected hippocampal tissues revealed hippocampal sclerosis with neuronal loss in the CA1 pyramidal layer, neuronal cell atrophy of the dentate gyrus, and thinning of the granule cell layer [42]. ...
... Thus, overexpression of NPY and Y2 receptors within the hippocampus may halt and reverse the frequency of status epilepticus. Exogenous NPY therapy may be an emerging gene therapy that may help reduce the epileptic activity after a cerebrovascular accident (Table 3) [42]. ...
Stroke is the most common cause of epilepsy and ultimately leads to a decrease in the quality of life of those affected. Ischemic and hemorrhagic strokes can both lead to poststroke epilepsy (PSE). Significant risk factors for PSE include age < 65 age less than 65 years, stroke severity measured by the National Institutes of Health Stroke Scale (NIHSS), cortical involvement, and genetic factors such as TRPM6 polymorphism. The diagnosis of PSE is made by using imaging modalities, blood biomarkers, and prognostic criteria. Electroencephalography (EEG) is currently the gold standard to diagnose PSE, while new combinations of modalities are being tested to increase diagnostic specificity. This literature review uncovers a newly found mechanism for the pathology of poststroke epilepsy. The pathogenesis of early-onset and late-onset is characterized by sequelae of neuronal cellular hypoxia and disruption of the blood-brain barrier, respectively. Interleukin-6 is responsible for increasing the activity of glial cells, causing gliosis and hyperexcitability of neurons. Epinephrine, high-mobility group protein B1, downregulation of CD32, and upregulation of HLA-DR impact the pathology of poststroke epilepsy by inhibiting the normal neuronal immune response. Decreased levels of neuropeptide Y, a neurotransmitter, act through multiple unique mechanisms, such as inhibiting intracellular Ca2+ accumulation and acting as an anti-inflammatory, also implemented in the worsening progression of poststroke epilepsy. Additionally, CA1 hippocampal resonant neurons that increase theta oscillation are associated with poststroke epilepsy. Hypertensive small vessel disease may also have an implication in the temporal lobe epilepsy by causing occult microinfarctions. Furthermore, this review highlights the potential use of statins as primary prophylaxis against PSE, with multiple studies demonstrating a reduction in incidence using statins alone, statins in combination with antiepileptic drugs (AEDs), and statins with aspirin. The evidence strongly suggests that the second generation AEDs are a superior treatment method for PSE. Data from numerous studies demonstrate their relative lack of significant drug interactions, increased tolerability, and potential superiority in maintaining seizure-free status.
... Temporal lobe epilepsy (TLE) is characterized by recurrent spontaneous seizures that originate in the temporal lobes, due to excessive synchronous neuronal firing (Wickham et al., 2019). In severe TLE, characteristic pathological changes in the DG include granule cell dispersion, GABAergic interneuron loss, and astrogliosis; a suite of histopathological changes referred to as hippocampal sclerosis (Mathern et al., 2002;Losi et al., 2012;Asadi-Pooya et al., 2017;Liu et al., 2020;Ammothumkandy et al., 2022). ...
GABAergic interneurons play a role in regulating adult neurogenesis within the dentate gyrus (DG) of the hippocampus. Neurogenesis occurs within a stem cell niche in the subgranular zone (SGZ) of the DG. In this niche, populations of neural progenitors give rise to granule cells that migrate radially into the granule cell layer of the DG. Altered neurogenesis in temporal lobe epilepsy (TLE) is linked to a transient increase in the proliferation of new neurons and the abnormal inversion of Type 1 progenitors, resulting in ectopic migration of Type 3 progenitors into the hilus of the DG. These ectopic cells mature into granule cells in the hilus that become hyperexcitable and contribute to the development of spontaneous recurrent seizures. To test whether grafts of GABAergic cells in the DG restore synaptic inhibition, prior work focused on transplanting GABAergic progenitors into the hilus of the DG. This cell-based therapeutic approach was shown to alter the disease phenotype by ameliorating spontaneous seizures in mice with pilocarpine-induced TLE. Prior optogenetic and immunohistochemical studies demonstrated that the transplanted GABAergic interneurons increased levels of synaptic inhibition by establishing inhibitory synaptic contacts with adult-born granule cells, consistent with the observed suppression of seizures. Whether GABAergic progenitor transplantation into the DG ameliorates underlying abnormalities in adult neurogenesis caused by TLE is not known. As a first step to address this question, we compared the effects of GABAergic progenitor transplantation on Type 1, Type 2, and Type 3 progenitors in the stem cell niche using cell type-specific molecular markers in naïve, non-epileptic mice. The progenitor transplantation increased GABAergic interneurons in the DG and led to a significant reduction in Type 2 progenitors and a concomitant increase in Type 3 progenitors. Next, we compared the effects of GABAergic interneuron transplantation in epileptic mice. Transplantation of GABAergic progenitors resulted in reductions in inverted Type 1, Type 2, and hilar ectopic Type 3 cells, concomitant with an increase in the radial migration of Type 3 progenitors into the GCL (Granule Cell Layer). Thus, in mice with Pilocarpine induced TLE, hilar transplants of GABA interneurons may reverse abnormal patterns of adult neurogenesis, an outcome that may ameliorate seizures.
... There are no data available in the accessible literature on the inhibition of postischemic excitotoxicity and/or PIDs after stimulation of Y2R. However, Y2R stimulation has been demonstrated to suppress excitotoxicity in epileptic attacks, both in experiments as well as in patients suffering from epilepsy [108][109][110][111][112]. The intracellular mechanisms connected with the suppression of neuronal excitability during the stimulation of Y2 receptors consist, in addition to the direct inhibition of cAMP/PKA signaling, of attenuation of the conductance of the N and P/Q calcium channels [78,80] and an increase in the conductance of the Kir channels [113]. ...
Ischemic stroke is the third leading cause of death and disability worldwide, with no available satisfactory prevention or treatment approach. The current treatment is limited to the use of “reperfusion methods,” i.e., an intravenous or intra-arterial infusion of a fibrinolytic agent, mechanical removal of the clot by thrombectomy, or a combination of both methods. It should be stressed, however, that only approximately 5% of all acute strokes are eligible for fibrinolytic treatment and fewer than 10% for thrombectomy. Despite the tremendous progress in understanding of the pathomechanisms of cerebral ischemia, the promising results of basic research on neuroprotection are not currently transferable to human stroke. A possible explanation for this failure is that experiments on in vivo animal models involve healthy young animals, and the experimental protocols seldom consider the importance of protecting the whole neurovascular unit (NVU), which ensures intracranial homeostasis and is seriously damaged by ischemia/reperfusion. One of the endogenous protective systems activated during ischemia and in neurodegenerative diseases is represented by neuropeptide Y (NPY). It has been demonstrated that activation of NPY Y2 receptors (Y2R) by a specific ligand decreases the volume of the postischemic infarction and improves performance in functional tests of rats with arterial hypertension subjected to middle cerebral artery occlusion/reperfusion. This functional improvement suggests the protection of the NVU. In this review, we focus on NPY and discuss the potential, multidirectional protective effects of Y2R agonists against acute focal ischemia/reperfusion injury, with special reference to the NVU.
... Increased hippocampal expression of NPY has been reported in many models of induced seizures, such as electrical kindling and kainic acid-induced models (83,84). Many studies have shown seizure-suppressing effects of NPY in the hippocampus of both rodents (84)(85)(86)(87)(88) and hippocampal slices from pharmacoresistant epilepsy patients (89)(90)(91). NPY elicits its biological actions in the brain mainly by binding to Y1, Y2, and Y5 receptors, members of a G-protein coupled receptor superfamily (92). In the hippocampus, the seizuresuppressing effects of NPY appear to be mediated primarily via activation of Y2 receptors (93), while Y5 receptors may also play a role particularly outside the hippocampus (86,87,94). ...
Temporal lobe epilepsy (TLE) is one of the most common and severe types of epilepsy, characterized by intractable, recurrent, and pharmacoresistant seizures. Histopathology of TLE is mostly investigated through observing hippocampal sclerosis (HS) in adults, which provides a robust means to analyze the related histopathological lesions. However, most pathological processes underlying the formation of these lesions remain elusive, as they are difficult to detect and observe. In recent years, significant efforts have been put in elucidating the pathophysiological pathways contributing to TLE epileptogenesis. In this review, we aimed to address the new and unrecognized neuropathological discoveries within the last 5 years, focusing on gene expression (miRNA and DNA methylation), neuronal peptides (neuropeptide Y), cellular metabolism (mitochondria and ion transport), cellular structure (microtubule and extracellular matrix), and tissue-level abnormalities (enlarged amygdala). Herein, we describe a range of biochemical mechanisms and their implication for epileptogenesis. Furthermore, we discuss their potential role as a target for TLE prevention and treatment. This review article summarizes the latest neuropathological discoveries at the molecular, cellular, and tissue levels involving both animal and patient studies, aiming to explore epileptogenesis and highlight new potential targets in the diagnosis and treatment of TLE.
... 160 Wickham et al. 152 demonstrate that NPY application, in hippocampal slices surgically resected from patients with drug-resistant TLE, significantly reduces chemically induced epileptiform activity in the dentate gyrus. 152 Increasing the levels of NPY could be an alternative approach to achieve a therapeutic effect and suppress seizure activity. ...
In recent years, precision medicine has emerged as a new paradigm for improved and more individualized patient care. Its key objective is to provide the right treatment, to the right patient at the right time, by basing medical decisions on individual characteristics, including specific genetic biomarkers. In order to realize this objective researchers and physicians must first identify the underlying genetic cause; over the last 10 years, advances in genetics have made this possible for several monogenic epilepsies. Through next generation techniques, a precise genetic aetiology is attainable in 30–50% of genetic epilepsies beginning in the paediatric age. While committed in such search for novel genes carrying disease‐causing variants, progress in the study of experimental models of epilepsy has also provided a better understanding of the mechanisms underlying the condition. Such advances are already being translated into improving care, management and treatment of some patients. Identification of a precise genetic aetiology can already direct physicians to prescribe treatments correcting specific metabolic defects, avoid antiseizure medicines that might aggravate functional consequences of the disease‐causing variant or select the drugs that counteract the underlying, genetically determined, functional disturbance. Personalized, tailored treatments should not just focus on how to stop seizures but possibly prevent their onset and cure the disorder, often consisting of seizures and its comorbidities including cognitive, motor and behaviour deficiencies. This review discusses the therapeutic implications following a specific genetic diagnosis and the correlation between genetic findings, pathophysiological mechanisms and tailored seizure treatment, emphasizing the impact on current clinical practice.
