In mammalian neurons, the axon initial segment (AIS) electrically connects the somatodendritic compartment with the axon and converts the incoming synaptic voltage changes into a temporally precise action potential (AP) output code. Although axons often emanate directly from the soma, they may also originate more distally from a dendrite, the implications of which are not wellunderstood. Here, we show that one-third of the thick-tufted layer 5 pyramidal neurons have an axon originating from a dendrite and are characterized by a reduced dendritic complexity and thinner main apical dendrite. Unexpectedly, the rising phase of somatic APs is electrically indistinguishable between neurons with a somatic or a dendritic axon origin. Cable analysis of the neurons indicated that the axonal axial current is inversely proportional to the AIS distance, denoting the path length between the soma and the start of the AIS, and to produce invariant somatic APs, it must scale with the local somatodendritic capacitance. In agreement, AIS distance inversely correlates with the apical dendrite diameter, and model simulations confirmed that the covariation suffices to normalize the somatic AP waveform. Therefore, in pyramidal neurons, the AIS location is finely tuned with the somatodendritic capacitive load, serving as a homeostatic regulation of the somatic AP in the face of diverse neuronal morphologies.T he axon initial segment (AIS) specifies in vertebrate neurons a single domain for the final integration of synaptic input and the initiation of action potentials (APs) (1, 2). To rapidly produce large inward and outward currents mediating the AP, the AIS contains a complex arrangement of cytoskeletal and transmembrane proteins clustering high densities of voltage-gated sodium (Nav) and potassium (Kv) channels in the axolemma (2-4). Although the composition of ion channels is critical for initiation and regulation of firing patterns, there are emerging insights that the AIS is not operating in isolation but is also subject to activity-dependent changes in size and location constrained by the local dendritic branch geometry and the passive cable properties (5-7). Experimental studies linking changes in AIS length and neuronal output showed that an increased length facilitates AP generation (6,8). In these cases, the net increased excitability is a logical consequence of the larger Nav conductance. However, predicting the impact of AIS location on neuronal output is more complex. Experimental studies showed that an activity-dependent distal shift of the AIS is associated with decreased AP output (5). In contrast, model simulations showed that shifting the AIS distally promotes excitability (9). One of the critical factors influencing AIS excitability is the large somatodendritic membrane area acting as current sink for sodium current generated in the AIS (10-12). In this view, a distal anatomical location of the AIS increases electrical compartmentalization and facilitates axonal AP generation. Indeed, the local depolarization in the...

Variations in the human Crumbs homolog-1 (CRB1) gene lead to an array of retinal dystrophies including early-onset of retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) in children. To investigate the physiological roles of CRB1 and CRB2 in retinal Müller glial cells, we analysed mouse retinas lacking both proteins in Müller glial cells. The peripheral retina showed a faster progression of dystrophy than the central retina. The central retina showed retinal folds, disruptions at the outer limiting membrane, protrusion of photoreceptor nuclei into the inner and outer segment layers, and ingression of photoreceptor nuclei into the photoreceptor synaptic layer. The peripheral retina showed a complete loss of the photoreceptor synapse layer, intermingling of photoreceptor nuclei within the inner nuclear layer and ectopic photoreceptor cells in the ganglion cell layer. Electroretinography showed severe attenuation of the scotopic a-wave at 1 month of age with responses below detection levels at 3-months of age. The double knockout mouse retinas mimicked a phenotype equivalent to a clinical LCA phenotype due to loss of CRB1. Localization of CRB1 and CRB2 in non-human primate retinas was analyzed at the ultrastructural level. We found that non-human primate CRB1 and CRB2 proteins localized to the subapical region adjacent to adherens junctions at the outer limiting membrane in Müller glial cells and photoreceptors. Our data suggest that loss of CRB2 in Müller glial cells aggravates the CRB1-associated RP-like phenotype towards an LCA-like phenotype.

Objective In metachromatic leukodystrophy, a lysosomal storage disorder due to decreased arylsulfatase A activity, hematopoietic stem cell transplantation may stop brain demyelination and allow remyelination, thereby halting white matter degeneration. This is the first study to define the effects and therapeutic mechanisms of hematopoietic stem cell transplantation on brain tissue of transplanted metachromatic leukodystrophy patients. Methods Autopsy brain tissue was obtained from eight (two transplanted and six nontransplanted) metachromatic leukodystrophy patients, and two age‐matched controls. We examined the presence of donor cells by immunohistochemistry and microscopy. In addition, we assessed myelin content, oligodendrocyte numbers, and macrophage phenotypes. An unpaired t‐test, linear regression or the nonparametric Mann–Whitney U‐test was performed to evaluate differences between the transplanted, nontransplanted, and control group. Results In brain tissue of transplanted patients, we found metabolically competent donor macrophages expressing arylsulfatase A distributed throughout the entire white matter. Compared to nontransplanted patients, these macrophages preferentially expressed markers of alternatively activated, anti‐inflammatory cells that may support oligodendrocyte survival and differentiation. Additionally, transplanted patients showed higher numbers of oligodendrocytes and evidence for remyelination. Contrary to the current hypothesis on therapeutic mechanism of hematopoietic cell transplantation in metachromatic leukodystrophy, we detected no enzymatic cross‐correction to resident astrocytes and oligodendrocytes. Interpretation In conclusion, donor macrophages are able to digest accumulated sulfatides and may play a neuroprotective role for resident oligodendrocytes, thereby enabling remyelination, albeit without evidence of cross‐correction of oligo‐ and astroglia. These results emphasize the importance of immunomodulation in addition to the metabolic correction, which might be exploited for improved outcomes.

SummaryEnsheathment of axons by myelin is a highly complex and multi-cellular process. Cytosolic calcium (Ca2+) changes in the myelin sheath have been implicated in myelin synthesis, but the source of this Ca2+ and the role of neuronal activity is not well understood. Using one-photon Ca2+ imaging, we investigated myelin sheath formation in the mouse somatosensory cortex and found a high rate of spontaneous microdomain Ca2+ transients and large-amplitude Ca2+ waves propagating along the internode. The frequency of Ca2+ transients and waves rapidly declines with maturation and reactivates during remyelination. Unexpectedly, myelin microdomain Ca2+ transients occur independent of neuronal action potential generation or network activity but are nearly completely abolished when the mitochondrial permeability transition pores are blocked. These findings are supported by the discovery of mitochondria organelles in non-compacted myelin. Together, the results suggest that myelin microdomain Ca2+ signals are cell-autonomously driven by high activity of mitochondria during myelin remodeling.

The complement system is implicated in synapse loss in the MS hippocampus, but the functional consequences of synapse loss remain poorly understood. Here, in post-mortem MS hippocampi with demyelination we find that deposits of the complement component C1q are enriched in the CA2 subfield, are linked to loss of inhibitory synapses and are significantly higher in MS patients with cognitive impairments compared to those with preserved cognitive functions. Using the cuprizone mouse model of demyelination, we corroborated that C1q deposits are highest within the demyelinated dorsal hippocampal CA2 pyramidal layer and co-localized with inhibitory synapses engulfed by microglia/macrophages. In agreement with the loss of inhibitory perisomatic synapses, we found that Schaffer collateral feedforward inhibition but not excitation was impaired in CA2 pyramidal neurons and accompanied by intrinsic changes and a reduced spike output. Finally, consistent with excitability deficits, we show that cuprizone-treated mice exhibit impaired encoding of social memories. Together, our findings identify CA2 as a critical circuit in demyelinated intrahippocampal lesions and memory dysfunctions in MS.

ObjectiveWe aimed to study the occurrence and development of axonal pathology and the influence of astrocytes in vanishing white matter.MethodsAxons and myelin were analyzed using electron microscopy and immunohistochemistry on Eif2b4 and Eif2b5 single‐ and double‐mutant mice and patient brain tissue. In addition, astrocyte‐forebrain co‐culture studies were performed.ResultsIn the corpus callosum of Eif2b5‐mutant mice, myelin sheath thickness, axonal diameter, and G‐ratio developed normally up to 4 months. At 7 months, however, axons had become thinner, while in control mice axonal diameters had increased further. Myelin sheath thickness remained close to normal, resulting in an abnormally low G‐ratio in Eif2b5‐mutant mice. In more severely affected Eif2b4‐Eif2b5 double‐mutants, similar abnormalities were already present at 4 months, while in milder affected Eif2b4 mutants, few abnormalities were observed at 7 months. Additionally, from 2 months onward an increased percentage of thin, unmyelinated axons and increased axonal density were present in Eif2b5‐mutant mice. Co‐cultures showed that Eif2b5 mutant astrocytes induced increased axonal density, also in control forebrain tissue, and that control astrocytes induced normal axonal density, also in mutant forebrain tissue. In vanishing white matter patient brains, axons and myelin sheaths were thinner than normal in moderately and severely affected white matter. In mutant mice and patients, signs of axonal transport defects and cytoskeletal abnormalities were minimal.InterpretationIn vanishing white matter, axons are initially normal and atrophy later. Astrocytes are central in this process. If therapy becomes available, axonal pathology may be prevented with early intervention.

Brain oedema is a life-threatening complication of various neurological conditions. Understanding molecular mechanisms of brain volume regulation is critical for therapy development. Unique insight comes from monogenic diseases characterized by chronic brain oedema, of which megalencephalic leukoencephalopathy with subcortical cysts (MLC) is the prototype. Variants in MLC1 or GLIALCAM, encoding proteins involved in astrocyte volume regulation, are the main causes of MLC. In some patients the genetic cause remains unknown. We performed genetic studies to identify novel gene variants in MLC patients, diagnosed by clinical and MRI features, without MLC1 or GLIALCAM variants. We determined subcellular localization of the related novel proteins in cells and in human brain tissue. We investigated functional consequences of the newly identified variants on volume regulation pathways using cell volume measurements, biochemical analysis and electrophysiology. We identified a novel homozygous variant in AQP4, encoding the water channel aquaporin-4, in two siblings, and two de novo heterozygous variants in GPRC5B, encoding the orphan G protein-coupled receptor GPRC5B, in three unrelated patients. The AQP4 variant disrupts membrane localization and thereby channel function. GPRC5B, like MLC1, GlialCAM and aquaporin-4, is expressed in astrocyte endfeet in human brain. Cell volume regulation is disrupted in GPRC5B patient-derived lymphoblasts. GPRC5B functionally interacts with ion channels involved in astrocyte volume regulation. In conclusion, we identify aquaporin-4 and GPRC5B as old and new players in genetic brain oedema. Our findings shed light on the protein complex involved in astrocyte volume regulation and identify GPRC5B as novel potentially druggable target for treating brain oedema.

The complement system is implicated in synapse loss in the MS hippocampus, but the functional consequences of synapse loss remain poorly understood. Here, in post-mortem MS hippocampi with demyelination we find that deposits of the complement component C1q are enriched in the CA2 subfield, are linked to loss of inhibitory synapses and are significantly higher in MS patients with cognitive impairments compared to those with preserved cognitive functions. Using the cuprizone mouse model of demyelination, we corroborated that C1q deposits are highest within the demyelinated dorsal hippocampal CA2 pyramidal layer, and co-localized with inhibitory synapses engulfed by microglia/macrophages. In agreement with the loss of inhibitory perisomatic synapses, we further found that Schaffer collateral feedforward inhibition but not excitation was impaired in CA2 pyramidal neurons and accompanied by a reduced spike output. Ultimately, we show that these electrophysiological changes were associated with an impaired encoding of social memories. Together, our findings identify CA2 as a critical circuit in demyelinated intrahippocampal lesions and memory dysfunctions in MS.