kcna1a mutant zebrafish model episodic ataxia type 1 (EA1) with epilepsy and show response to first-line therapy carbamazepine.

OBJECTIVE: KCNA1 mutations are associated with a rare neurological movement disorder known as episodic ataxia type 1 (EA1), and epilepsy is a common comorbidity. Current medications provide only partial relief for ataxia and/or seizures, making new drugs needed. Here, we characterized zebrafish kcna1a-/- as a model of EA1 with epilepsy and compared the efficacy of the first-line therapy carbamazepine in kcna1a-/- zebrafish to Kcna1-/- rodents. METHODS: CRISPR/Cas9 mutagenesis was used to introduce a mutation in the sixth transmembrane segment of the zebrafish Kcna1 protein. Behavioral and electrophysiological assays were performed on kcna1a-/- larvae to assess ataxia- and epilepsy-related phenotypes. Real-time quantitative polymerase chain reaction (qPCR) was conducted to measure mRNA levels of brain hyperexcitability markers in kcna1a-/- larvae, followed by bioenergetics profiling to evaluate metabolic function. Drug efficacies were tested using behavioral and electrophysiological assessments, as well as seizure frequency in kcna1a-/- zebrafish and Kcna1-/- mice, respectively. RESULTS: Zebrafish kcna1a-/- larvae showed uncoordinated movements and locomotor deficits, along with scoliosis and increased mortality. The mutants also exhibited impaired startle responses when exposed to light-dark flashes and acoustic stimulation as well as hyperexcitability as measured by extracellular field recordings and upregulated fosab transcripts. Neural vglut2a and gad1b transcript levels were disrupted in kcna1a-/- larvae, indicative of a neuronal excitatory/inhibitory imbalance, as well as a significant reduction in cellular respiration in kcna1a-/- , consistent with dysregulation of neurometabolism. Notably, carbamazepine suppressed the impaired startle response and brain hyperexcitability in kcna1a-/- zebrafish but had no effect on the seizure frequency in Kcna1-/- mice, suggesting that this EA1 zebrafish model might better translate to humans than rodents. SIGNIFICANCE: We conclude that zebrafish kcna1a-/- show ataxia and epilepsy-related phenotypes and are responsive to carbamazepine treatment, consistent with EA1 patients. These findings suggest that kcna1-/- zebrafish are a useful model for drug screening as well as studying the underlying disease biology.

A novel metabolism-based phenotypic drug discovery platform in zebrafish uncovers HDACs 1 and 3 as a potential combined anti-seizure drug target.

Despite the development of newer anti-seizure medications over the past 50 years, 30-40% of patients with epilepsy remain refractory to treatment. One explanation for this lack of progress is that the current screening process is largely biased towards transmembrane channels and receptors, and ignores intracellular proteins and enzymes that might serve as efficacious molecular targets. Here, we report the development of a novel drug screening platform that harnesses the power of zebrafish genetics and combines it with in vivo bioenergetics screening assays to uncover therapeutic agents that improve mitochondrial health in diseased animals. By screening commercially available chemical libraries of approved drugs, for which the molecular targets and pathways are well characterized, we were able to reverse-identify the proteins targeted by efficacious compounds and confirm the physiological roles that they play by utilizing other pharmacological ligands. Indeed, using an 870-compound screen in kcna1-morpholino epileptic zebrafish larvae, we uncovered vorinostat (Zolinza™; suberanilohydroxamic acid, SAHA) as a potent anti-seizure agent. We further demonstrated that vorinostat decreased average daily seizures by ∼60% in epileptic Kcna1-null mice using video-EEG recordings. Given that vorinostat is a broad histone deacetylase (HDAC) inhibitor, we then delineated a specific subset of HDACs, namely HDACs 1 and 3, as potential drug targets for future screening. In summary, we have developed a novel phenotypic, metabolism-based experimental therapeutics platform that can be used to identify new molecular targets for future drug discovery in epilepsy.

A model of chronic diabetic polyneuropathy: benefits from intranasal insulin are modified by sex and RAGE deletion.

Human diabetic polyneuropathy (DPN) is a progressive complication of chronic diabetes mellitus. Preliminary evidence has suggested that intranasal insulin, in doses insufficient to alter hyperglycemia, suppresses the development of DPN. In this work we confirm this finding, but demonstrate that its impact is modified by sex and deletion of RAGE, the receptor for advanced glycosylation end products. We serially evaluated experimental DPN in male and female wild-type mice and male RAGE null (RN) mice, each with nondiabetic controls, during 16 wk of diabetes, the final 8 wk including groups given intranasal insulin. Age-matched nondiabetic female mice had higher motor and sensory conduction velocities than their male counterparts and had lesser conduction slowing from chronic diabetes. Intranasal insulin improved slowing in both sexes. In male RN mice, there was less conduction slowing with chronic diabetes, and intranasal insulin provided limited benefits. Rotarod testing and hindpaw grip power offered less consistent impacts. Mechanical sensitivity and thermal sensitivity were respectively but disparately changed and improved with insulin in wild-type female and male mice but not RN male mice. These studies confirm that intranasal insulin improves indexes of experimental DPN but indicates that females with DPN may differ in their underlying phenotype. RN mice had partial but incomplete protection from underlying DPN and lesser impacts from insulin. We also identify an important role for sex in the development of DPN and report evidence that insulin and AGE-RAGE pathways in its pathogenesis may overlap.

Axo-glial communication through neurexin-neuroligin signaling regulates myelination and oligodendrocyte differentiation.

Axonal transsynaptic signaling between presynaptic neurexin (NX) and postsynaptic neuroligin (NL) is essential for many properties of synaptic connectivity. Here, we demonstrate the existence of a parallel axo-glial signaling pathway between axonal NX and oligodendritic (OL) NL3. We show that this pathway contributes to the regulation of myelinogenesis, the maintenance of established myelination, and the differentiation state of the OL using in vitro models. We first confirm that NL3 mRNA and protein are expressed in OLs and in OL precursors. We then show that OLs in culture form contacts with non-neuronal cells exogenously expressing NL3's binding partners NX1α or NX1ß. Conversely, blocking axo-glial NX-NL3 signaling by saturating NX with exogenous soluble NL protein (NL-ECD) disrupts interactions between OLs and axons in both in vitro and ex vivo assays. Myelination by OLs is tied to their differentiation state, and we find that blocking NX-NL signaling with soluble NL protein also caused OL differentiation to stall at an immature stage. Moreover, in vitro knockdown of NL3 in OLs with siRNAs stalls their development and reduces branching complexity. Interestingly, inclusion of an autism related mutation in the NL blocking protein attenuates these effects; OLs differentiate and the dynamics of OL-axon signaling occur normally as this peptide does not disrupt NX-NL3 axo-glial interactions. Our findings provide evidence not only for a new pathway in axo-glial communication, they also potentially explain the correlation between altered white matter and autism. GLIA 2015;63:2023-2039.

Sudden unexpected death in epilepsy is prevented by blocking postictal hypoxia.

Epilepsy is at times a fatal disease. Sudden unexpected death in epilepsy (SUDEP) is the leading cause of epilepsy-related mortality in people with intractable epilepsy and is defined by exclusion; non-accidental, non-toxicologic, and non-anatomic causes of death. While SUDEP often follows a bilateral tonic-clonic seizure, the mechanisms that ultimately lead to terminal apnea and then asystole remain elusive and there is a lack of preventative treatments. Based on the observation that discrete seizures lead to local and postictal vasoconstriction, resulting in hypoperfusion, hypoxia and behavioural disturbances in the forebrain we reasoned those similar mechanisms may play a role in SUDEP when seizures invade the brainstem. Here we tested this neurovascular-based hypothesis of SUDEP in awake non-anesthetized mice by pharmacologically preventing seizure-induced vasoconstriction, with cyclooxygenase-2 or L-type calcium channel antagonists. In both acute and chronic mouse models of seizure-induced premature mortality, ibuprofen and nicardipine extended life while systemic drug levels remained high enough to be effective. We also examined the potential role of spreading depolarization in the acute model of seizure-induced premature mortality. These data provide a proof-of-principle for the neurovascular hypothesis of SUDEP rather than spreading depolarization and the use of currently available drugs to prevent it.

Distribution of inducible nitric oxide synthase and tumor necrosis factor-alpha in the peripheral nervous system of Lewis rats during ascending paresis and spontaneous recovery from experimental autoimmune neuritis.

BACKGROUND: Inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-alpha) are pleiotropic molecules with widespread action in autoimmune diseases. OBJECTIVE: This study characterizes the distribution of iNOS and TNF-alpha in the spinal nerve roots, dorsal root ganglia and sciatic nerve of Lewis rats during experimental autoimmune neuritis (EAN). METHODS: Macrophages and neutrophils were identified by immunofluorescence as cellular sources of iNOS and TNF-alpha at various stages of EAN induced by synthetic peptide 26. RESULTS: As the disease progressed, iNOS- and TNF-alpha-bearing cells gradually infiltrated the cauda equina, dorsal root ganglia, Th12-L3 spinal roots, and the sciatic nerve. A severer EAN profile developed when more iNOS- and TNF-alpha-bearing cells were present, and the recovery from EAN was related to the disappearance of these cells and the regeneration of nerve fibers. CONCLUSIONS: This is the first report to show iNOS- and TNF-alpha-immunoreactive cells in dorsal root ganglia during EAN, suggesting an underlying pathology for the neuropathic pain behavior in EAN. Our results suggest that the cells bearing iNOS and TNF-alpha in the different parts of the peripheral nervous system are involved in the development of the clinical signs observed at each stage of EAN.

Maturity of the myenteric plexus is decreased in the gastroschisis rat model.

BACKGROUND: Amniotic fluid (AF) and its components, such as fetal urine and meconium, may lead to intestinal alterations in gastroschisis, which cause immaturity of the myenteric plexus and consequent intestinal hypomotility and malabsorption. In this study we identified morphological and histological alterations of the intestine and the myenteric plexus with two different times of exposure to AF. METHODS: The experimental gastroschisis was achieved at two different gestational ages, on day 18.5 (E18.5) and day 19.5 (E19.5) of gestation, in fetal rats which were divided into 3 subgroups: control, sham and gastroschisis. We measured fetal body weight (BW), intestinal weight (IW) and intestinal length (IL). The layers of intestinal wall and myenteric plexus were evaluated by hematoxylin and eosin staining (HE staining) and immunofluorescence (alpha-internexin), respectively. RESULTS: BW was not significantly different among the control, sham and gastroschisis groups at both ages. IW and IL were larger and shorter, respectively, in the gastroschisis fetuses (p < 0.001) at both ages. Intestinal diameters and wall layers presented significant differences among control, sham and gastroschisis fetuses at both ages (p < 0.001), but the time of exposure to AF compromised the serous membrane, D-II (diameter II, p < 0.001) and IL (p = 0.001). alpha-Internexin presented more intensive immunoreactivity in gastroschisis fetuses at E18.5. CONCLUSIONS: In gastroschisis, the longer the time of exposure to AF, the more severe bowel impairment will be, especially with regard to IL and the serous layer, and the more immature the myenteric plexus will be.

The effect of treatment with crotapotin on the evolution of experimental autoimmune neuritis induced in Lewis rats.

Biomedical research in which venom components are being investigated for their potential as novel therapeutic agents has emerged as an interesting option. Crotapotin, which is purified from the venom of the rattlesnake Crotalus durissus terrificus, has been described as an anti-inflammatory agent that acts on the innate arm of the immune response. Here we have demonstrated that intraperitoneal administration of crotapotin significantly reduces the severity of experimental autoimmune neuritis (EAN), an experimental model for Guillain-Barré syndrome. The reduction of the severity of the disease is associated with a reduction in the mononuclear cells infiltrating the sciatic nerve and a significant decrease in the lymphocyte proliferative response to neuritogenic peptide.

Diabetic polyneuropathy, sensory neurons, nuclear structure and spliceosome alterations: a role for CWC22.

Unique deficits in the function of adult sensory neurons as part of their early neurodegeneration might account for progressive polyneuropathy during chronic diabetes mellitus. Here, we provide structural and functional evidence for aberrant pre-mRNA splicing in a chronic type 1 model of experimental diabetic polyneuropathy (DPN). Cajal bodies (CBs), unique nuclear substructures involved in RNA splicing, increased in number in diabetic sensory neurons, but their expected colocalization with survival motor neuron (SMN) proteins was reduced - a mislocalization described in motor neurons of spinal muscular atrophy. Small nuclear ribonucleoprotein particles (snRNPs), also participants in the spliceosome, had abnormal multiple nuclear foci unassociated with CBs, and their associated snRNAs were reduced. CWC22, a key spliceosome protein, was aberrantly upregulated in diabetic dorsal root ganglia (DRG), and impaired neuronal function. CWC22 attenuated sensory neuron plasticity, with knockdown in vitro enhancing their neurite outgrowth. Further, axonal delivery of CWC22 siRNA unilaterally to locally knock down the aberrant protein in diabetic nerves improved aspects of sensory function in diabetic mice. Collectively, our findings identify subtle but significant alterations in spliceosome structure and function, including dysregulated CBs and CWC22 overexpression, in diabetic sensory neurons that offer new ideas regarding diabetic sensory neurodegeneration in polyneuropathy.

Changes in MMPs and inflammatory cells in experimental gingivitis.

In periodontal disease, extensive disorganization of the extracellular matrix promotes the loss of adhesion between the teeth and periodontium. A previous study suggested a reduction in the area occupied by collagen in the gingiva, during the first week of periodontal disease induction, however, the remaining fibers were more compact and thicker. Therefore, it was decided to investigate which of the MMP-2, -9, -14 and RECK, an MMP inhibitor, were involved in these modifications taking place in early gingivitis induced by ligature. The results of gene expression analysis indicated no changes for RECK. MMP-14 showed a reduction at 7 days of inflammation, and there was an immediate increase in MMP-2 gene expression and enzymatic activity, apparently by the stimulation of resident cells such as fibroblasts. A peak of MMP-9 expression 5 days after ligature followed after the peak of enzymatic activity found two days earlier. This pattern was consistent with the kinetics of macrophage and neutrophil recruitment. Immunohistochemistry suggested that MMP-9 was produced by both resident and inflammatory cells. Based on this evidence, it is suggested that extracellular matrix remodeling is related to MMP-2 and -9 production and activation. This allowed us to conclude that the host inflammatory response represents a significant factor for the advance of periodontal diseases.

Nitric oxide and TNFalpha effects in experimental autoimmune encephalomyelitis demyelination.

The involvement of inducible nitric oxide synthase (iNOS), which plays various roles in the progression of autoimmune diseases, was studied in iNOS knockout (KO) mice and wild-type (WT) controls with respect to experimental autoimmune encephalomyelitis (EAE). The iNOS (KO) mice presented a less severe form of the disease than the WT control mice. Although the levels of TNFalpha decreased in the periphery in both groups, an increase in the number of TNFalpha-positive cells was detected in the central nervous system during the acute phase of EAE in the WT mice, but not in the KO mice. These findings suggest that NO and TNFalpha contribute to the pathogenesis of acute EAE.

Wallerian degeneration in C57BL/6J and A/J mice: differences in time course of neurofilament and myelin breakdown, macrophage recruitment and iNOS expression.

The lower regeneration potential reported for C57BL/6J mice strain after peripheral nerve lesion may result from alterations in crucial events during Wallerian degeneration. We analysed neurofilament and myelin breakdown, macrophage recruitment, NADPH-diaphorase reaction and inducible nitric oxide synthase (iNOS) expression in transected sciatic nerves of C57BL/6J and A/J mice. The neurofilament volume density was lower in C57BL/6J strain mice at 1 and 3 days after lesion, and later equalled the density observed in A/J. C57BL/6J mice presented a high number of cells containing myelin debris, 3 and 5 days after the lesion. In both strains iNOS immunoreactivity was intense in macrophages and less evident in Schwann cells. However, a delay in macrophage recruitment and a lower percentage of iNOS-expressing macrophages on the third day were observed in C57BL/6J mice. NADPH-diaphorase reaction disclosed a similar pattern for both strains until the seventh day. However, at 5 days, cells with slender processes involving ellipsoid segments showed a well-defined cytoplasmic labelling in C57BL/6J whereas in A/J most of these cells exhibited a more granular and disperse labelling. We propose that these differences between A/J and C57BL/6J strains during Wallerian degeneration may be implicated in the lower regeneration potential observed in the latter.