Steven S. Scherer

A Common Ankyrin-G-Based Mechanism Retains KCNQ and NaV Channels at Electrically Active Domains of the Axon

KCNQ (KV7) potassium channels underlie subthreshold M-currents that stabilize the neuronal resting potential and prevent repetitive firing of action potentials. Here, antibodies against four different KCNQ2 and KCNQ3 polypeptide epitopes show these subunits concentrated at the axonal initial segment (AIS) and node of Ranvier. AIS concentration of KCNQ2 and KCNQ3, like that of voltage-gated sodium (NaV) channels, is abolished in ankyrin-G knock-out mice. A short motif, common to KCNQ2 and KCNQ3, mediates both in vivo ankyrin-G interaction and retention of the subunits at the AIS. This KCNQ2/KCNQ3 motif is nearly identical to the sequence on NaV α subunits that serves these functions. All identified NaV and KCNQ genes of worms, insects, and molluscs lack the ankyrin-G binding motif. In contrast, vertebrate orthologs of NaV α subunits, KCNQ2, and KCNQ3 (including from bony fish, birds, and mammals) all possess the motif. Thus, concerted ankyrin-G interaction with KCNQ and NaV channels appears to have arisen through convergent molecular evolution, after the division between invertebrate and vertebrate lineages, but before the appearance of the last common jawed vertebrate ancestor. This includes the historical period when myelin also evolved.

KCNQ2 Is a Nodal K+ Channel

Mutations in the gene encoding the K+ channel KCNQ2 cause neonatal epilepsy and myokymia, indicating that KCNQ2 regulates the excitability of CNS neurons and motor axons, respectively. We show here that KCNQ2 channels are functional components of axon initial segments and nodes of Ranvier, colocalizing with ankyrin-G and voltage-dependent Na+ channels throughout the CNS and PNS. Retigabine, which opens KCNQ channels, diminishes axonal excitability. Linopirdine, which blocks KCNQ channels, prolongs the repolarization of the action potential in neonatal nerves. The clustering of KCNQ2 at nodes and initial segments lags that of ankyrin-G during development, and both ankyrin-G and KCNQ2 can be coimmunoprecipitated in the brain. KCNQ3 is also a component of some initial segments and nodes in the brain. The diminished activity of mutant KCNQ2 channels accounts for neonatal epilepsy and myokymia; the cellular locus of these effects may be axonal initial segments and nodes.

Disease mechanisms in inherited neuropathies

Inherited neuropathies are caused by dominant or recessive mutations in genes that are expressed by neurons and/or Schwann cells. In demyelinating neuropathies, the deleterious effects originate primarily in myelinating Schwann cells. In axonal neuropathies, neurons (axons) are initially affected. In demyelinating neuropathies, the axonal cytoskeleton is altered and axonal transport is disrupted. In some axonal neuropathies, genes that are directly involved in axonal transport are mutated. So, a common consequence of inherited neuropathies is disruption of the ability of neurons to transport cargo efficiently along the entire length of their axons. These findings correlate with the observations that axonal atrophy and/or loss are primarily responsible for the clinical disability in hereditary neuropathies.

Regulation of ciliary neurotrophic factor expression in myelin-related Schwann cells in vivo

Adult rat sciatic nerve is known to express high levels of ciliary neurotrophic factor (CNTF) mRNA and protein. Here we examine the cellular localization of CNTF protein and mRNA in peripheral nerve and the regulation of CNTF expression by peripheral axons. In intact nerve, CNTF immunoreactivity is found predominantly in the cytoplasm of myelin-related Schwann cells. After axotomy, CNTF immunoreactivity and mRNA levels fall dramatically and do not recover unless axons regenerate. This behavior is similar to the pattern of myelin gene expression in these nerves. We conclude that the expression of CNTF in Schwann cells depends on axon-Schwann cell interactions.

Connexin32‐null mice develop demyelinating peripheral neuropathy

Mutations in the gene encoding the gap junction protein connexin32 (Cx32) cause X‐linked Charcot‐Marie‐Tooth disease (CMTX), a common form of inherited demyelinating peripheral neuropathy. To learn more about the pathogenesis of CMTX, we examined the PNS and CNS of cx32‐null mice (cx32−/Y males and cx32−/− females) by light and electron microscopy. These mice develop a progressive demyelinating peripheral neuropathy beginning by 3 months of age, and at all ages, motor fibers are more affected than sensory fibers. Like other genes of the X chromosome, the cx32 gene appears to be randomly inactivated, since only some myelinating Schwann cells express Cx32 in heterozygous cx32+/− females. Heterozygous cx32+/− females have fewer demyelinated and remyelinated axons than age‐matched homozygous cx32−/− females and cx32−/Y males. Although oligodendrocytes also express Cx32, no abnormalities in CNS myelin were found. These findings indicate that a null cx32 allele in myelinating Schwann cells is sufficient to cause an inherited demyelinating neuropathy, so that Cx32 has an essential role in myelinating Schwann cells both in mice and in humans. GLIA 24:8–20, 1998.

Retrograde axonal transport of LIF is increased by peripheral nerve injury: Correlation with increased LIF expression in distal nerve

Leukemia inhibitory factor (LIF) is a cytokine that affects the survival and differentiation of certain neuronal populations in vitro. To identify LIF-responsive neurons in the adult rat, we have demonstrated retrograde axonal transport of 125I-LIF to sensory and motor neurons. The accumulation of 125I-LIF by both cell types was significantly increased by prior sciatic nerve crush. Retrograde transport of 125I-LIF was inhibited by excess unlabeled LIF but not by related cytokines, indicating a specific receptor-mediated mechanism. Northern blot analysis revealed LIF expression in peripheral nerve that was increased in distal segments after axotomy. The correlation between LIF expression and increased retrograde transport following injury suggests that LIF plays a role in peripheral nerve regeneration.

Investigations of Caspr2, an autoantigen of encephalitis and neuromyotonia

To report clinical and immunological investigations of contactin‐associated protein‐like 2 (Caspr2), an autoantigen of encephalitis and peripheral nerve hyperexcitability (PNH) previously attributed to voltage‐gated potassium channels (VGKC).

On the molecular architecture of myelinated fibers.

Abstract Schwann cells and oligodendrocytes make the myelin sheaths of the PNS and CNS, respectively. Their myelin sheaths are structurally similar, consisting of multiple layers of specialized cell membrane that spiral around axons, but there are several differences. (1) CNS myelin has a ”radial component” composed of a tight junction protein, claudin-11/oligodendrocyte-specific protein. (2) Schwann cells have a basal lamina and microvilli. (3) Although both CNS and PNS myelin sheaths have incisures, those in the CNS lack the structural as well as the molecular components of ”reflexive” adherens junctions and gap junctions. In spite of their structural differences, the axonal membranes of the PNS and CNS are similarly organized. The nodal axolemma contains high concentrations of voltage-dependent sodium channels that are linked to the axonal cytoskeleton by ankyrinG. The paranodal membrane contains Caspr/paranodin, which may participate in the formation of axoglial junctions. The juxtaparanodal axonal membrane contains the potassium channels Kv1.1 and Kv1.2, their associated β2 subunit, as well as Caspr2, which is closely related to Caspr. The myelin sheath probably organizes these axonal membrane-related proteins via trans interactions.

Null mutations of connexin32 in patients with X-linked Charcot-Marie-Tooth disease

The X-linked form of Charcot-Marie-Tooth disease (CMTX) is associated with mutations in the gene encoding connexin32, a member of the family of proteins forming intercellular channels. We have compared the functional properties of three mutant connexin32 genes with those of the wild-type gene by testing their ability to form intercellular channels in the paired oocyte expression system. Whereas wild-type connexin32 induced the development of large junctional conductance between paired oocytes, no functional channels were detected between pairs expressing CMTX mutants. Furthermore, CMTX mutants selectively acted as dominant inhibitors of intercellular communication by interfering with the channel-forming ability of connexin26 but not with that of connexin40. These results demonstrate a functional loss in the product of a candidate gene for a demyelinating form of CMT.

A central role for Necl4 (SynCAM4) in Schwann cell–axon interaction and myelination

Myelination in the peripheral nervous system requires close contact between Schwann cells and the axon, but the underlying molecular basis remains largely unknown. Here we show that cell adhesion molecules (CAMs) of the nectin-like (Necl, also known as SynCAM or Cadm) family mediate Schwann cell–axon interaction during myelination. Necl4 is the main Necl expressed by myelinating Schwann cells and is located along the internodes in direct apposition to Necl1, which is localized on axons. Necl4 serves as the glial binding partner for axonal Necl1, and the interaction between these two CAMs mediates Schwann cell adhesion. The disruption of the interaction between Necl1 and Necl4 by their soluble extracellular domains, or the expression of a dominant-negative Necl4 in Schwann cells, inhibits myelination. These results suggest that Necl proteins are important for mediating axon-glia contact during myelination in peripheral nerves.