Central nervous system hypomyelination disrupts axonal conduction and behaviour in larval zebrafish

Myelination is essential for central nervous system (CNS) formation, health and function. As a model organism, larval zebrafish have been extensively employed to investigate the molecular and cellular basis of CNS myelination, due to their genetic tractability and suitability for non-invasive live cell imaging. However, it has not been assessed to what extent CNS myelination affects neural circuit function in zebrafish larvae, prohibiting the integration of molecular and cellular analyses of myelination with concomitant network maturation. To test whether larval zebrafish might serve as a suitable platform with which to study the effects of CNS myelination and its dysregulation on circuit function, we generated zebrafish myelin regulatory factor (myrf) mutants with CNS-specific hypomyelination and investigated how this affected their axonal conduction properties and behaviour. We found that myrf mutant larvae exhibited increased latency to perform startle responses following defined acoustic stimuli. Furthermore, we found that hypomyelinated animals often selected an impaired response to acoustic stimuli, exhibiting a bias towards reorientation behaviour instead of the stimulus-appropriate startle response. To begin to study how myelination affected the underlying circuitry, we established electrophysiological protocols to assess various conduction properties along single axons. We found that the hypomyelinated myrf mutants exhibited reduced action potential conduction velocity and an impaired ability to sustain high frequency action potential firing. This study indicates that larval zebrafish can be used to bridge molecular and cellular investigation of CNS myelination with multiscale assessment of neural circuit function.SIGNIFICANCE STATEMENTMyelination of central nervous system axons is essential for their health and function, and it now clear that myelination is a dynamic life-long process subject to modulation by neuronal activity. However, it remains unclear precisely how changes to myelination affects animal behaviour and underlying action potential conduction along axons in intact neural circuits. In recent years, zebrafish have been employed to study cellular and molecular mechanisms of myelination, due to their relatively simple, optically transparent, experimentally tractable vertebrate nervous system. Here we find that changes to myelination alter the behaviour of young zebrafish and action potential conduction along individual axons, providing a platform to integrate molecular, cellular and circuit level analyses of myelination using this model

CNS Hypomyelination Disrupts Axonal Conduction and Behavior in Larval Zebrafish

Myelination is essential for central nervous system (CNS) formation, health and function. As a model organism, larval zebrafish have been extensively employed to investigate the molecular and cellular basis of CNS myelination, because of their genetic tractability and suitability for non-invasive live cell imaging. However, it has not been assessed to what extent CNS myelination affects neural circuit function in zebrafish larvae, prohibiting the integration of molecular and cellular analyses of myelination with concomitant network maturation. To test whether larval zebrafish might serve as a suitable platform with which to study the effects of CNS myelination and its dysregulation on circuit function, we generated zebrafish myelin regulatory factor (myrf) mutants with CNS-specific hypomyelination and investigated how this affected their axonal conduction properties and behavior. We found that myrf mutant larvae exhibited increased latency to perform startle responses following defined acoustic stimuli. Furthermore, we found that hypomyelinated animals often selected an impaired response to acoustic stimuli, exhibiting a bias toward reorientation behavior instead of the stimulus-appropriate startle response. To begin to study how myelination affected the underlying circuitry, we established electrophysiological protocols to assess various conduction properties along single axons. We found that the hypomyelinated myrf mutants exhibited reduced action potential conduction velocity and an impaired ability to sustain high-frequency action potential firing. This study indicates that larval zebrafish can be used to bridge molecular and cellular investigation of CNS myelination with multiscale assessment of neural circuit function. SIGNIFICANCE STATEMENT Myelination of CNS axons is essential for their health and function, and it is now clear that myelination is a dynamic life-long process subject to modulation by neuronal activity. However, it remains unclear precisely how changes to myelination affects animal behavior and underlying action potential conduction along axons in intact neural circuits. In recent years, zebrafish have been employed to study cellular and molecular mechanisms of myelination, because of their relatively simple, optically transparent, experimentally tractable vertebrate nervous system. Here we find that changes to myelination alter the behavior of young zebrafish and action potential conduction along individual axons, providing a platform to integrate molecular, cellular, and circuit level analyses of myelination using this model

Importin 13-dependent axon diameter growth regulates conduction speeds along myelinated CNS axons

Axon diameter influences the conduction properties of myelinated axons, both directly, and indirectly through effects on myelin. However, we have limited understanding of mechanisms controlling axon diameter growth in the central nervous system, preventing systematic dissection of how manipulating diameter affects myelination and conduction along individual axons. Here we establish zebrafish to study axon diameter. We find that importin 13b is required for axon diameter growth, but does not affect cell body size or axon length. Using neuron-specific ipo13b mutants, we assess how reduced axon diameter affects myelination and conduction, and find no changes to myelin thickness, precision of action potential propagation, or ability to sustain high frequency firing. However, increases in conduction speed that occur along single myelinated axons with development are tightly linked to their growth in diameter. This suggests that axon diameter growth is a major driver of increases in conduction speeds along myelinated axons over time.<br/

Neuronal activity disrupts myelinated axon integrity in the absence of NKCC1b

Through a genetic screen in zebrafish, we identified a mutant with disruption to myelin in both the CNS and PNS caused by a mutation in a previously uncharacterized gene, slc12a2b, predicted to encode a Na+, K+, and Cl− (NKCC) cotransporter, NKCC1b. slc12a2b/NKCC1b mutants exhibited a severe and progressive pathology in the PNS, characterized by dysmyelination and swelling of the periaxonal space at the axon–myelin interface. Cell-type–specific loss of slc12a2b/NKCC1b in either neurons or myelinating Schwann cells recapitulated these pathologies. Given that NKCC1 is critical for ion homeostasis, we asked whether the disruption to myelinated axons in slc12a2b/NKCC1b mutants is affected by neuronal activity. Strikingly, we found that blocking neuronal activity completely prevented and could even rescue the pathology in slc12a2b/NKCC1b mutants. Together, our data indicate that NKCC1b is required to maintain neuronal activity–related solute homeostasis at the axon–myelin interface, and the integrity of myelinated axons

Endothelin signalling mediates experience-dependent myelination in the CNS

Experience and changes in neuronal activity can alter CNS myelination, but the signalling pathways responsible remain poorly understood. Here we define a pathway in which endothelin, signalling through the G protein-coupled receptor endothelin receptor B and PKC epsilon, regulates the number of myelin sheaths formed by individual oligodendrocytes in mouse and zebrafish. We show that this phenotype is also observed in the prefrontal cortex of mice following social isolation, and is associated with reduced expression of vascular endothelin. Additionally, we show that increasing endothelin signalling rescues this myelination defect caused by social isolation. Together, these results indicate that the vasculature responds to changes in neuronal activity associated with experience by regulating endothelin levels, which in turn affect the myelinating capacity of oligodendrocytes. This pathway may be employed to couple the metabolic support function of myelin to activity-dependent demand and also represents a novel mechanism for adaptive myelination

Altered network properties in C9ORF72 repeat expansion cortical neurons are due to synaptic dysfunction

Background Physiological disturbances in cortical network excitability and plasticity are established and widespread in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients, including those harbouring the C9ORF72 repeat expansion (C9ORF72RE) mutation – the most common genetic impairment causal to ALS and FTD. Noting that perturbations in cortical function are evidenced pre-symptomatically, and that the cortex is associated with widespread pathology, cortical dysfunction is thought to be an early driver of neurodegenerative disease progression. However, our understanding of how altered network function manifests at the cellular and molecular level is not clear. Methods To address this we have generated cortical neurons from patient-derived iPSCs harbouring C9ORF72RE mutations, as well as from their isogenic expansion-corrected controls. We have established a model of network activity in these neurons using multi-electrode array electrophysiology. We have then mechanistically examined the physiological processes underpinning network dysfunction using a combination of patch-clamp electrophysiology, immunocytochemistry, pharmacology and transcriptomic profiling. Results We find that C9ORF72RE causes elevated network burst activity, associated with enhanced synaptic input, yet lower burst duration, attributable to impaired pre-synaptic vesicle dynamics. We also show that the C9ORF72RE is associated with impaired synaptic plasticity. Moreover, RNA-seq analysis revealed dysregulated molecular pathways impacting on synaptic function. All molecular, cellular and network deficits are rescued by CRISPR/Cas9 correction of C9ORF72RE. Our study provides a mechanistic view of the early dysregulated processes that underpin cortical network dysfunction in ALS-FTD. Conclusion These findings suggest synaptic pathophysiology is widespread in ALS-FTD and has an early and fundamental role in driving altered network function that is thought to contribute to neurodegenerative processes in these patients. The overall importance is the identification of previously unidentified defects in pre and postsynaptic compartments affecting synaptic plasticity, synaptic vesicle stores, and network propagation, which directly impact upon cortical function

Recessive mutations in SPTBN2 implicate β-III spectrin in both cognitive and motor development

β-III spectrin is present in the brain and is known to be important in the function of the cerebellum. Heterozygous mutations in SPTBN2, the gene encoding β-III spectrin, cause Spinocerebellar Ataxia Type 5 (SCA5), an adult-onset, slowly progressive, autosomal-dominant pure cerebellar ataxia. SCA5 is sometimes known as "Lincoln ataxia," because the largest known family is descended from relatives of the United States President Abraham Lincoln. Using targeted capture and next-generation sequencing, we identified a homozygous stop codon in SPTBN2 in a consanguineous family in which childhood developmental ataxia co-segregates with cognitive impairment. The cognitive impairment could result from mutations in a second gene, but further analysis using whole-genome sequencing combined with SNP array analysis did not reveal any evidence of other mutations. We also examined a mouse knockout of β-III spectrin in which ataxia and progressive degeneration of cerebellar Purkinje cells has been previously reported and found morphological abnormalities in neurons from prefrontal cortex and deficits in object recognition tasks, consistent with the human cognitive phenotype. These data provide the first evidence that β-III spectrin plays an important role in cortical brain development and cognition, in addition to its function in the cerebellum; and we conclude that cognitive impairment is an integral part of this novel recessive ataxic syndrome, Spectrin-associated Autosomal Recessive Cerebellar Ataxia type 1 (SPARCA1). In addition, the identification of SPARCA1 and normal heterozygous carriers of the stop codon in SPTBN2 provides insights into the mechanism of molecular dominance in SCA5 and demonstrates that the cell-specific repertoire of spectrin subunits underlies a novel group of disorders, the neuronal spectrinopathies, which includes SCA5, SPARCA1, and a form of West syndrome

Cerebellar ataxias: β-III spectrin’s interactions suggest common pathogenic pathways.

Spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of disorders all characterised by postural abnormalities, motor deficits and cerebellar degeneration. Animal and in vitro models have revealed β‐III spectrin, a cytoskeletal protein present throughout the soma and dendritic tree of cerebellar Purkinje cells, to be required for the maintenance of dendritic architecture and for the trafficking and/or stabilisation of several membrane proteins: ankyrin‐R, cell adhesion molecules, metabotropic glutamate receptor‐1 (mGluR1), voltage‐gated sodium channels (Na(v)) and glutamate transporters. This scaffold of interactions connects β‐III spectrin to a wide variety of proteins implicated in the pathology of many SCAs. Heterozygous mutations in the gene encoding β‐III spectrin (SPTBN2) underlie SCA type‐5 whereas homozygous mutations cause spectrin associated autosomal recessive ataxia type‐1 (SPARCA1), an infantile form of ataxia with cognitive impairment. Loss‐of β‐III spectrin function appears to underpin cerebellar dysfunction and degeneration in both diseases resulting in thinner dendrites, excessive dendritic protrusion with loss of planarity, reduced resurgent sodium currents and abnormal glutamatergic neurotransmission. The initial physiological consequences are a decrease in spontaneous activity and excessive excitation, likely to be offsetting each other, but eventually hyperexcitability gives rise to dark cell degeneration and reduced cerebellar output. Similar molecular mechanisms have been implicated for SCA1, 2, 3, 7, 13, 14, 19, 22, 27 and 28, highlighting alterations to intrinsic Purkinje cell activity, dendritic architecture and glutamatergic transmission as possible common mechanisms downstream of various loss‐of‐function primary genetic defects. A key question for future research is whether similar mechanisms underlie progressive cerebellar decline in normal ageing. [Image: see text