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.

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.

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.

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.