Neurofilament accumulations in amyotrophic lateral sclerosis patients' motor neurons impair axonal initial segment integrity.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron (MN) disease in adults with no curative treatment. Neurofilament (NF) level in patient' fluids have recently emerged as the prime biomarker of ALS disease progression, while NF accumulation in MNs of patients is the oldest and one of the best pathological hallmarks. However, the way NF accumulations could lead to MN degeneration remains unknown. To assess NF accumulations and study the impact on MNs, we compared MNs derived from induced pluripotent stem cells (iPSC) of patients carrying mutations in C9orf72, SOD1 and TARDBP genes, the three main ALS genetic causes. We show that in all mutant MNs, light NF (NF-L) chains rapidly accumulate in MN soma, while the phosphorylated heavy/medium NF (pNF-M/H) chains pile up in axonal proximal regions of only C9orf72 and SOD1 MNs. Excitability abnormalities were also only observed in these latter MNs. We demonstrate that the integrity of the MN axonal initial segment (AIS), the region of action potential initiation and responsible for maintaining axonal integrity, is impaired in the presence of pNF-M/H accumulations in C9orf72 and SOD1 MNs. We establish a strong correlation between these pNF-M/H accumulations, an AIS distal shift, increased axonal calibers and modified repartition of sodium channels. The results expand our understanding of how NF accumulation could dysregulate components of the axonal cytoskeleton and disrupt MN homeostasis. With recent cumulative evidence that AIS alterations are implicated in different brain diseases, preserving AIS integrity could have important therapeutic implications for ALS.

Variants in the SK2 channel gene (KCNN2) lead to dominant neurodevelopmental movement disorders.

KCNN2 encodes the small conductance calcium-activated potassium channel 2 (SK2). Rodent models with spontaneous Kcnn2 mutations show abnormal gait and locomotor activity, tremor and memory deficits, but human disorders related to KCNN2 variants are largely unknown. Using exome sequencing, we identified a de novo KCNN2 frameshift deletion in a patient with learning disabilities, cerebellar ataxia and white matter abnormalities on brain MRI. This discovery prompted us to collect data from nine additional patients with de novo KCNN2 variants (one nonsense, one splice site, six missense variants and one in-frame deletion) and one family with a missense variant inherited from the affected mother. We investigated the functional impact of six selected variants on SK2 channel function using the patch-clamp technique. All variants tested but one, which was reclassified to uncertain significance, led to a loss-of-function of SK2 channels. Patients with KCNN2 variants had motor and language developmental delay, intellectual disability often associated with early-onset movement disorders comprising cerebellar ataxia and/or extrapyramidal symptoms. Altogether, our findings provide evidence that heterozygous variants, likely causing a haploinsufficiency of the KCNN2 gene, lead to novel autosomal dominant neurodevelopmental movement disorders mirroring phenotypes previously described in rodents.

New role of P2X7 receptor in an Alzheimer's disease mouse model.

Extracellular aggregates of amyloid ß (Aß) peptides, which are characteristic of Alzheimer's disease (AD), act as an essential trigger for glial cell activation and the release of ATP, leading to the stimulation of purinergic receptors, especially the P2X7 receptor (P2X7R). However, the involvement of P2X7R in the development of AD is still ill-defined regarding the dual properties of this receptor. Particularly, P2X7R activates the NLRP3 inflammasome leading to the release of the pro-inflammatory cytokine, IL-1ß; however, P2X7R also induces cleavage of the amyloid precursor protein generating Aß peptides or the neuroprotective fragment sAPPα. We thus explored in detail the functions of P2X7R in AD transgenic mice. Here, we show that P2X7R deficiency reduced Aß lesions, rescued cognitive deficits and improved synaptic plasticity in AD mice. However, the lack of P2X7R did not significantly affect the release of IL-1ß or the levels of non-amyloidogenic fragment, sAPPα, in AD mice. Instead, our results show that P2X7R plays a critical role in Aß peptide-mediated release of chemokines, particularly CCL3, which is associated with pathogenic CD8+ T cell recruitment. In conclusion, our study highlights a novel detrimental function of P2X7R in chemokine release and supports the notion that P2X7R may be a promising therapeutic target for AD.

HCN1 mutation spectrum: from neonatal epileptic encephalopathy to benign generalized epilepsy and beyond.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels control neuronal excitability and their dysfunction has been linked to epileptogenesis but few individuals with neurological disorders related to variants altering HCN channels have been reported so far. In 2014, we described five individuals with epileptic encephalopathy due to de novo HCN1 variants. To delineate HCN1-related disorders and investigate genotype-phenotype correlations further, we assembled a cohort of 33 unpublished patients with novel pathogenic or likely pathogenic variants: 19 probands carrying 14 different de novo mutations and four families with dominantly inherited variants segregating with epilepsy in 14 individuals, but not penetrant in six additional individuals. Sporadic patients had epilepsy with median onset at age 7 months and in 36% the first seizure occurred during a febrile illness. Overall, considering familial and sporadic patients, the predominant phenotypes were mild, including genetic generalized epilepsies and genetic epilepsy with febrile seizures plus (GEFS+) spectrum. About 20% manifested neonatal/infantile onset otherwise unclassified epileptic encephalopathy. The study also included eight patients with variants of unknown significance: one adopted patient had two HCN1 variants, four probands had intellectual disability without seizures, and three individuals had missense variants inherited from an asymptomatic parent. Of the 18 novel pathogenic missense variants identified, 12 were associated with severe phenotypes and clustered within or close to transmembrane domains, while variants segregating with milder phenotypes were located outside transmembrane domains, in the intracellular N- and C-terminal parts of the channel. Five recurrent variants were associated with similar phenotypes. Using whole-cell patch-clamp, we showed that the impact of 12 selected variants ranged from complete loss-of-function to significant shifts in activation kinetics and/or voltage dependence. Functional analysis of three different substitutions altering Gly391 revealed that these variants had different consequences on channel biophysical properties. The Gly391Asp variant, associated with the most severe, neonatal phenotype, also had the most severe impact on channel function. Molecular dynamics simulation on channel structure showed that homotetramers were not conducting ions because the permeation path was blocked by cation(s) strongly complexed to the Asp residue, whereas heterotetramers showed an instantaneous current component possibly linked to deformation of the channel pore. In conclusion, our results considerably expand the clinical spectrum related to HCN1 variants to include common generalized epilepsy phenotypes and further illustrate how HCN1 has a pivotal function in brain development and control of neuronal excitability.

The endoplasmic reticulum-mitochondria interface is perturbed in PARK2 knockout mice and patients with PARK2 mutations.

Mutations in PARK2, encoding the E3 ubiquitin protein ligase Parkin, are a common cause of autosomal recessive Parkinson's disease (PD). Loss of PARK2 function compromises mitochondrial quality by affecting mitochondrial biogenesis, bioenergetics, dynamics, transport and turnover. We investigated the impact of PARK2 dysfunction on the endoplasmic reticulum (ER)-mitochondria interface, which mediates calcium (Ca2+) exchange between the two compartments and is essential for Parkin-dependent mitophagy. Confocal and electron microscopy analyses showed the ER and mitochondria to be in closer proximity in primary fibroblasts from PARK2 knockout (KO) mice and PD patients with PARK2 mutations than in controls. Ca2+ flux to the cytosol was also modified, due to enhanced ER-to-mitochondria Ca2+ transfers, a change that was also observed in neurons derived from induced pluripotent stem cells of a patient with PARK2 mutations. Subcellular fractionation showed the abundance of the Parkin substrate mitofusin 2 (Mfn2), which is known to modulate the ER-mitochondria interface, to be specifically higher in the mitochondrion-associated ER membrane compartment in PARK2 KO tissue. Mfn2 downregulation or the exogenous expression of normal Parkin restored cytosolic Ca2+ transients in fibroblasts from patients with PARK2 mutations. In contrast, a catalytically inactive PD-related Parkin variant had no effect. Overall, our data suggest that Parkin is directly involved in regulating ER-mitochondria contacts and provide new insight into the role of the loss of Parkin function in PD development.

C9ORF72 knockdown triggers FTD-like symptoms and cell pathology in mice.

The GGGGCC intronic repeat expansion within C9ORF72 is the most common genetic cause of ALS and FTD. This mutation results in toxic gain of function through accumulation of expanded RNA foci and aggregation of abnormally translated dipeptide repeat proteins, as well as loss of function due to impaired transcription of C9ORF72. A number of in vivo and in vitro models of gain and loss of function effects have suggested that both mechanisms synergize to cause the disease. However, the contribution of the loss of function mechanism remains poorly understood. We have generated C9ORF72 knockdown mice to mimic C9-FTD/ALS patients haploinsufficiency and investigate the role of this loss of function in the pathogenesis. We found that decreasing C9ORF72 leads to anomalies of the autophagy/lysosomal pathway, cytoplasmic accumulation of TDP-43 and decreased synaptic density in the cortex. Knockdown mice also developed FTD-like behavioral deficits and mild motor phenotypes at a later stage. These findings show that C9ORF72 partial loss of function contributes to the damaging events leading to C9-FTD/ALS.

The chimeric approach reveals that differences in the TRPV1 pore domain determine species-specific sensitivity to block of heat activation.

The capsaicin-, heat-, and proton-activated ion channel TRPV1, a member of the transient receptor potential cation channel family is a polymodal nociceptor. For almost a decade, TRPV1 has been explored by the pharmaceutical industry as a potential target for example for pain conditions. Antagonists which block TRPV1 activation by capsaicin, heat, and protons were developed by a number of pharmaceutical companies. The unexpected finding of hyperthermia as an on-target side effect in clinical studies using polymodal TRPV1 antagonists has prompted companies to search for ways to circumvent hyperthermia, for example by the development of modality-selective antagonists. The significant lack of consistency of the pharmacology of many TRPV1 antagonists across different species has been a further obstacle. JYL-1421 for example was shown to block capsaicin and heat responses in human and monkey TRPV1 while it was largely ineffective in blocking heat responses in rat TRPV1. These findings suggested structural dissimilarities between different TRPV1 species relevant for small compound antagonism for example of heat activation. Using a chimeric approach (human and rat TRPV1) in combination with a novel FLIPR-based heat activation assay and patch-clamp electrophysiology we have identified the pore region as being strongly linked to the observed species differences. We demonstrate that by exchanging the pore domains JYL-1421, which is modality-selective in rat can be made modality-selective in human TRPV1 and vice-versa.

Systematic analysis and prediction of genes associated with monogenic disorders on human chromosome X.

Disease gene discovery on chromosome (chr) X is challenging owing to its unique modes of inheritance. We undertook a systematic analysis of human chrX genes. We observe a higher proportion of disorder-associated genes and an enrichment of genes involved in cognition, language, and seizures on chrX compared to autosomes. We analyze gene constraints, exon and promoter conservation, expression, and paralogues, and report 127 genes sharing one or more attributes with known chrX disorder genes. Using machine learning classifiers trained to distinguish disease-associated from dispensable genes, we classify 247 genes, including 115 of the 127, as having high probability of being disease-associated. We provide evidence of an excess of variants in predicted genes in existing databases. Finally, we report damaging variants in CDK16 and TRPC5 in patients with intellectual disability or autism spectrum disorders. This study predicts large-scale gene-disease associations that could be used for prioritization of X-linked pathogenic variants.

Transcriptional control of KCNQ channel genes and the regulation of neuronal excitability.

Regulation of the resting membrane potential and the repolarization of neurons are important in regulating neuronal excitability. The potassium channel subunits Kv7.2 and Kv7.3 play a key role in stabilizing neuronal activity. Mutations in KCNQ2 and KCNQ3, the genes encoding Kv7.2 and Kv7.3, cause a neonatal form of epilepsy, and activators of these channels have been identified as novel antiepileptics and analgesics. Despite the observations that regulation of these subunits has profound effects on neuronal function, almost nothing is known about the mechanisms responsible for controlling appropriate expression levels. Here we identify two mechanisms responsible for regulating KCNQ2 and KCNQ3 mRNA levels. We show that the transcription factor Sp1 activates expression of both KCNQ2 and KCNQ3, whereas the transcriptional repressor REST (repressor element 1-silencing transcription factor) represses expression of both of these genes. Furthermore, we show that transcriptional regulation of KCNQ genes is mirrored by the correlated changes in M-current density and excitability of native sensory neurons. We propose that these mechanisms are important in the control of excitability of neurons and may have implications in seizure activity and pain.

3-O-sulfated heparan sulfate interactors target synaptic adhesion molecules from neonatal mouse brain and inhibit neural activity and synaptogenesis in vitro.

Heparan sulfate (HS) chains, covalently linked to heparan sulfate proteoglycans (HSPG), promote synaptic development and functions by connecting various synaptic adhesion proteins (AP). HS binding to AP could vary according to modifications of HS chains by different sulfotransferases. 3-O-sulfotransferases (Hs3sts) produce rare 3-O-sulfated HSs (3S-HSs), of poorly known functions in the nervous system. Here, we showed that a peptide known to block herpes simplex virus by interfering with 3S-HSs in vitro and in vivo (i.e. G2 peptide), specifically inhibited neural activity, reduced evoked glutamate release, and impaired synaptic assembly in hippocampal cell cultures. A role for 3S-HSs in promoting synaptic assembly and neural activity is consistent with the synaptic interactome of G2 peptide, and with the detection of Hs3sts and their products in synapses of cultured neurons and in synaptosomes prepared from developing brains. Our study suggests that 3S-HSs acting as receptors for herpesviruses might be important regulators of neuronal and synaptic development in vertebrates.

A204E mutation in Nav1.4 DIS3 exerts gain- and loss-of-function effects that lead to periodic paralysis combining hyper- with hypo-kalaemic signs.

Periodic paralyses (PP) are characterized by episodic muscle weakness and are classified into the distinct hyperkalaemic (hyperPP) and hypokalaemic (hypoPP) forms. The dominantly-inherited form of hyperPP is caused by overactivity of Nav1.4 - the skeletal muscle voltage-gated sodium channel. Familial hypoPP results from a leaking gating pore current induced by dominant mutations in Nav1.4 or Cav1.1, the skeletal muscle voltage-gated calcium channel. Here, we report an individual with clinical signs of hyperPP and hypokalaemic episodes of muscle paralysis who was heterozygous for the novel p.Ala204Glu (A204E) substitution located in one region of Nav1.4 poor in disease-related variations. A204E induced a significant decrease of sodium current density, increased the window current, enhanced fast and slow inactivation of Nav1.4, and did not cause gating pore current in functional analyses. Interestingly, the negative impact of A204E on Nav1.4 activation was strengthened in low concentration of extracellular K+. Our data prove the existence of a phenotype combining signs of hyperPP and hypoPP due to dominant Nav1.4 mutations. The hyperPP component would result from gain-of-function effects on Nav1.4 and the hypokalemic episodes of paralysis from loss-of-function effects strengthened by low K+. Our data argue for a non-negligible role of Nav1.4 loss-of-function in familial hypoPP.

A case of non-dystrophic myotonia with concomitant mutations in the SCN4A and CLCN1 genes.

Non-dystrophic myotonias are caused by mutations of either the skeletal muscle chloride (CLCN1) or sodium channel (SCN4A) gene. They exhibit several distinct phenotypes, including myotonia congenita, paramyotonia congenita and sodium channel myotonia, and a genotype-phenotype correlation has been established. However, there are atypical cases that do not fit with the standard classification. We report a case of 27-year-old male who had non-dystrophic myotonia with periodic paralysis and two heterozygous mutations, E950K in CLCN1 and F1290L in SCN4A. His mother, who exhibited myotonia without paralytic attack, only harbored E950K, and no mutations were identified in his asymptomatic father. Therefore, the E950K mutation was presumed to be pathogenic, although it was reported as an extremely rare genetic variant. The proband experienced paralytic attacks that lasted for weeks and were less likely to be caused by CLCN1 mutation alone. Functional analysis of the F1290L mutant channel heterologously expressed in cultured cells revealed enhanced activation inducing membrane hyperexcitability. We therefore propose that the two mutations had additive effects on membrane excitability that resulted in more prominent myotonia in the proband. Our case stresses the value of performing genetic analysis of both CLCN1 and SCN4A genes for myotonic patients with an atypical phenotype.

Peripheral block of the hyperpolarization-activated cation current (Ih) reduces mechanical allodynia in animal models of postoperative and neuropathic pain.

BACKGROUND AND OBJECTIVES: Block of the hyperpolarization-activated inward current (I h) reduces excitability of peripheral axons during stimulation and decreases ectopic discharges in axotomized sensory neurons. Changes in I h expression in DRG neurons have been suggested to partially underlie sensitization after nerve injury and inflammation. We hypothesized that peripheral block of I h on axons would produce an antiallodynic effect in postoperative as well as neuropathic conditions, and we tested perineural administration of ZD 7288, a specific blocker of I h , on pain-associated behavior in animal models of neuropathic and postoperative pain. METHODS: Under halothane anesthesia, partial sciatic nerve injury or hind-paw incision were performed on adult male rats as previously described. Mechanical allodynia was inferred by demonstration of a decrease in paw withdrawal threshold by application of calibrated von Frey filaments. After surgery, animals received either a saline or a ZD 7288 solution either by sciatic perineural injection or by intraplantar injection. RESULTS: Perineural administration of ZD 7288 (100 microM) significantly reduced mechanical allodynia induced by partial sciatic nerve injury and hind-paw incision. Saline and 10 microM of ZD 7288 had no significant effect on mechanical allodynia. Contralateral administration of ZD 7288, 100 microM, did not affect ipsilateral paw withdrawal threshold after nerve injury. Intraplantar injection of ZD 7288 failed to reduce mechanical allodynia after nerve injury. Sedation and motor effects were not observed. CONCLUSIONS: The current study shows that peripheral block of I h produces an antiallodynic effect, which suggests that I h channels represent a novel target for nerve block treatment of postoperative and neuropathic pain.

Axon-glia interactions modulate axonal excitability in mammalian unmyelinated nerves.

A period of electrical activity in unmyelinated nerve fibers is followed by a post-tetanic hyperpolarization (PTH), generated by the hyperactivity of the electrogenic Na(+)-K(+) pump. In order to protect the membrane potential against these strong hyperpolarizations, different types of axonal inward currents are activated during the PTH. We investigated in the rabbit vagus nerve one of these currents, which was activated by carbamylcholine (CCh). We observed that the effect of CCh on the PTH amplitude could be blocked or reversed with scopolamine. Moreover, the PTH amplitude increased when scopolamine alone was added to the perfusate, indicating that an endogenous muscarinic agonist was liberated in the preparation during the period of electrical activity. This CCh-activated current was TEA but not Ba(2+) or Cs(+) sensitive. It has been shown previously that muscarinic acetylcholine receptors (mAChRs) in the rabbit vagus nerve are located on the axonal but not glial membrane and that Schwann cells express several types of purinergic receptors, which activation evoke Ca(2+) transients in Schwann cells. We hypothesise that during electrical activity axons release a transmitter, presumably ATP. This transmitter evoke in the neighbouring Schwann cells a Ca(2+)-dependent liberation of a endogenous muscarinic agonist, which in turn activates a TEA-sensitive inward current in axons. We suggest that the major purpose of this mechanism is the control of the membrane potential during and after a period of intense electrical activity when the Na(+)-K(+) pump generates a robust PTH.

De novo mutations in HCN1 cause early infantile epileptic encephalopathy.

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels contribute to cationic Ih current in neurons and regulate the excitability of neuronal networks. Studies in rat models have shown that the Hcn1 gene has a key role in epilepsy, but clinical evidence implicating HCN1 mutations in human epilepsy is lacking. We carried out exome sequencing for parent-offspring trios with fever-sensitive, intractable epileptic encephalopathy, leading to the discovery of two de novo missense HCN1 mutations. Screening of follow-up cohorts comprising 157 cases in total identified 4 additional amino acid substitutions. Patch-clamp recordings of Ih currents in cells expressing wild-type or mutant human HCN1 channels showed that the mutations had striking but divergent effects on homomeric channels. Individuals with mutations had clinical features resembling those of Dravet syndrome with progression toward atypical absences, intellectual disability and autistic traits. These findings provide clear evidence that de novo HCN1 point mutations cause a recognizable early-onset epileptic encephalopathy in humans.

Transcriptional repression of the M channel subunit Kv7.2 in chronic nerve injury.

Neuropathic pain is a severe health problem for which there is a lack of effective therapy. A frequent underlying condition of neuropathic pain is a sustained overexcitability of pain-sensing (nociceptive) sensory fibres. Therefore, the identification of mechanisms for such abnormal neuronal excitability is of utmost importance for understanding neuropathic pain. Despite much effort, an inclusive model explaining peripheral overexcitability is missing. We investigated transcriptional regulation of the Kcnq2 gene, which encodes the Kv7.2 subunit of membrane potential-stabilizing M channel, in peripheral sensory neurons in a model of neuropathic pain-partial sciatic nerve ligation (PSNL). We show that Kcnq2 is the major Kcnq gene transcript in dorsal root ganglion (DRG); immunostaining and patch-clamp recordings from acute ganglionic slices verified functional expression of Kv7.2 in small-diameter nociceptive DRG neurons. Neuropathic injury induced substantial downregulation of Kv7.2 expression. Levels of repressor element 1-silencing transcription factor (REST), which is known to suppress Kcnq2 expression, were upregulated in response to neuropathic injury identifying the likely mechanism of Kcnq2 regulation. Behavioural experiments demonstrated that neuropathic hyperalgesia following PSNL developed faster than the downregulation of Kcnq2 expression could be detected, suggesting that this transcriptional mechanism may contribute to the maintenance rather than the initiation of neuropathic pain. Importantly, the decrease in the peripheral M channel abundance could be functionally compensated by peripherally applied M channel opener flupirtine, which alleviated neuropathic hyperalgesia. Our work suggests a novel mechanism for neuropathic overexcitability and brings focus on M channels and REST as peripheral targets for the treatment of neuropathic pain.