Purpose: Several factors contribute to epileptogenesis in patients with brain tumors, including reduced γ-aminobutyric acid (GABA)ergic inhibition. In particular, changes in Cl⁻ homeostasis in peritumoral microenvironment, together with alterations of metabolism, are key processes leading to epileptogenesis in patients afflicted by glioma. It has been recently proposed that alterations of Cl⁻ homeostasis could be involved in tumor cell migration and metastasis formation. In neurons, the regulation of intracellular Cl⁻ concentration ([Cl⁻]_i) is mediated by NKCC1 and KCC2 transporters: NKCC1 increases while KCC2 decreases [Cl⁻]_i. Experiments were thus designed to investigate whether, in human epileptic peritumoral cortex, alterations in the balance of NKCC1 and KCC2 activity may decrease the hyperpolarizing effects of GABA, thereby contributing to epileptogenesis in human brain tumors. Methods: Membranes from peritumoral cortical tissues of epileptic patients afflicted by gliomas (from II to IV WHO grade) and from cortical tissues of nonepileptic patients were injected into Xenopus oocytes leading to the incorporation of functional GABA_A receptors. The GABA-evoked currents were recorded using standard two-microelectrode voltage-clamp technique. In addition, immunoblot analysis and immunohistochemical staining were carried out on membranes and tissues from the same patients. Key Findings: We found that in oocytes injected with epileptic peritumoral cerebral cortex, the GABA-evoked currents had a more depolarized reversal potential (E_GABA) compared to those from nonepileptic healthy cortex. This difference of E_GABA was abolished by the NKCC1 blocker bumetanide or unblocking of KCC2 with the Zn²⁺ chelator TPEN. Moreover, Western blot analysis revealed an increased expression of NKCC1, and more modestly, of KCC2 transporters in epileptic peritumoral tissues compared to nonepileptic control tissues. In addition, NKCC1 immunoreactivity was strongly increased in peritumoral cortex with respect to nonepileptic cortex, with a prominent expression in neuronal cells. Significance: We report that the positive shift of E_GABA in epileptic peritumoral human cortex is due to an altered expression of NKCC1 and KCC2, perturbing Cl⁻ homeostasis, which might lead to a consequent reduction in GABAergic inhibition. These findings point to a key role of Cl⁻ transporters KCC2 and NKCC1 in tumor-related epilepsy, suggesting a more specific drug therapy and surgical approaches for the epileptic patients afflicted by brain tumors.

Summary: Purpose: The chemokine fractalkine/CX3CL1 and its receptor CX3CR1 are widely expressed in the central nervous system (CNS). Recent evidence showed that CX3CL1 participates in inflammatory responses that are common features of CNS disorders, such as epilepsy. Mesial temporal lobe epilepsy (MTLE) is the prevalent form of focal epilepsy in adults, and hippocampal sclerosis (HS) represents the most common underlying pathologic abnormality, as demonstrated at autopsy and postresection studies. Relevant features of MTLE are a characteristic pattern of neuronal loss, as are astrogliosis and microglia activation. Several factors affect epileptogenesis in patients with MTLE, including a lack of γ‐aminobutyric acid (GABA)ergic inhibitory efficacy. Therefore, experiments were designed to investigate whether, in MTLE brain tissues, CX3CL1 may influence GABA_A receptor (GABA_AR) mediatedtransmission, with a particular focus on the action of CX3CL1 on the use‐dependent decrease (rundown) of the GABA‐evoked currents (I_GABA), a feature underlying the reduction of GABAergic function in epileptic tissue.Methods: Patch‐clamp recordings were obtained from cortical pyramidal neurons in slices from six MTLE patients after surgery. Alternatively, the cell membranes from epileptic brain tissues of 17 MTLE patients or from surgical samples and autopsies of nonepileptic patients were microtransplanted into Xenopus oocytes, and I_GABA were recorded using the standard two‐microelectrode voltage‐clamp technique. Immunohistochemical staining and double‐labeling studies were carried out on the same brain tissues to analyze CX3CR1 expression.Key Findings: In native pyramidal neurons from cortical slices of patients with MTLE, CX3CL1 reduced I_GABA rundown and affected the recovery of I_GABA amplitude from rundown. These same effects were confirmed in oocytes injected with cortical and hippocampal MTLE membranes, whereas CX3CL1 did not influence I_GABA in oocytes injected with nonepileptic tissues. Consistent with a specific effect of CX3CL1 on tissues from patients with MTLE, CX3CR1 immunoreactivity was higher in MTLE sclerotic hippocampi than in control tissues, with a prominent expression in activated microglial cells.Significance: These findings indicate a role for CX3CL1 in MTLE, supporting recent evidence on the relevance of brain inflammation in human epilepsies. Our data demonstrate that in MTLE tissues the reduced GABAergic function can be modulated by CX3CL1. The increased CX3CR1 expression in microglia and the modulation by CX3CL1 of GABAergic currents in human epileptic brain suggests new therapeutic approaches for drug‐resistant epilepsies based on the evidence that the propagation of seizures can be influenced by inflammatory processes.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting motor neurons that leads to progressive paralysis of skeletal muscle. Studies of ALS have revealed defects in expression of acetylcholine receptors (AChRs) in skeletal muscle that occur even in the absence of motor neuron anomalies. The endocannabinoid palmitoylethanolamide (PEA) modified the clinical conditions in one ALS patient, improving muscle force and respiratory efficacy. By microtransplanting muscle membranes from selected ALS patients into Xenopus oocytes, we show that PEA reduces the desensitization of acetylcholine-evoked currents after repetitive neurotransmitter application (i.e., rundown). The same effect was observed using muscle samples from denervated (non-ALS) control patients. The expression of human recombinant α1β1γδ (γ-AChRs) and α1β1εδ AChRs (ε-AChRs) in Xenopus oocytes revealed that PEA selectively affected the rundown of ACh currents in ε-AChRs. A clear up-regulation of the α1 subunit in muscle from ALS patients compared with that from non-ALS patients was found by quantitative PCR, but no differential expression was found for other subunits. Clinically, ALS patients treated with PEA showed a lower decrease in their forced vital capacity (FVC) over time as compared with untreated ALS patients, suggesting that PEA can enhance pulmonary function in ALS. In the present work, data were collected from a cohort of 76 ALS patients and 17 denervated patients. Our results strengthen the evidence for the role of skeletal muscle in ALS pathogenesis and pave the way for the development of new drugs to hamper the clinical effects of the disease. Significance Amyotrophic lateral sclerosis (ALS) is a fatal disease leading to motor neuron degeneration and progressive paralysis. Other studies have revealed defects in skeletal muscle even in the absence of motor neuron anomalies, focusing on acetylcholine receptors (AChRs) and supporting the so-called “dying-back” hypothesis. Our results indicate that the endocannabinoid palmitoylethanolamide (PEA) reduces the rundown of AChRs currents in ALS muscle and can clinically improve patients’ pulmonary function. This study strengthens the important role of muscle in ALS pathogenesis and the idea that AChRs can be therapeutic targets.