Mollie Burgoon Woodworth

Summary Both cell-intrinsic competency and extracellular cues regulate axon projection, but mechanisms that coordinate these elements remain poorly understood. Subcerebral projection neurons (SCPN) extend their primary axons from cortex through subcortical structures, including the striatum, targeting the brainstem and spinal cord. We identify that the transcription factor Bcl11b/Ctip2 functions in multiple independent neuron populations to control SCPN axon development. Bcl11b expressed by SCPN is required cell-autonomously for axonal outgrowth and efficient entry into the internal capsule within the striatum, while Bcl11b expressed by medium spiny neurons (MSN) non-cell-autonomously regulates SCPN axon fasciculation within the internal capsule and subsequent pathfinding. Further, integrated investigation of Bcl11b-null SCPN with transcriptomic, immunocytochemical, and in vivo growth cone purification approaches identifies that Cdh13 is localized along axons and on growth cone surfaces of SCPN in vivo, and mediates Bcl11b regulation of SCPN axonal outgrowth. Together, these results demonstrate that Bcl11b controls multiple aspects of SCPN axon development by coordinating intrinsic SCPN cell autonomous subcellular mechanisms and extrinsic MSN non-cell-autonomous mechanisms.

SUMMARY: Both cell-intrinsic competency and extracellular cues regulate axon projection, but mechanisms that coordinate these elements remain poorly understood. Subcerebral projection neurons (SCPN) extend their primary axons from cortex through subcortical structures, including the striatum, targeting the brainstem and spinal cord. We identify that the transcription factor Bcl11b/Ctip2 functions in multiple independent neuron populations to control SCPN axon development. Bcl11b expressed by SCPN is required cell-autonomously for axonal outgrowth and efficient entry into the internal capsule within the striatum, while Bcl11b expressed by medium spiny neurons (MSN) non-cell-autonomously regulates SCPN axon fasciculation within the internal capsule and subsequent pathfinding. Further, integrated investigation of Bcl11b -null SCPN with transcriptomic, immunocytochemical, and in vivo growth cone purification approaches identifies that Cdh13 is localized along axons and on growth cone surfaces of SCPN in vivo , and mediates Bcl11b regulation of SCPN axonal outgrowth. Together, these results demonstrate that Bcl11b controls multiple aspects of SCPN axon development by coordinating intrinsic SCPN cell autonomous subcellular mechanisms and extrinsic MSN non-cell-autonomous mechanisms.

The intrinsic mechanisms that regulate neurotoxic versus neuroprotective astrocyte phenotypes and their effects on central nervous system (CNS) degeneration and repair remain poorly understood. Here, we show injured white matter astrocytes differentiate into two distinct C3-positive and C3-negative reactive populations, previously simplified as neurotoxic (A1) and neuroprotective (A2)^1,2, which can be further subdivided into unique subpopulations defined by proliferation and differential gene expression signatures. We find the balance of neurotoxic versus neuroprotective astrocytes is regulated by discrete pools of compartmented cAMP derived from soluble adenylyl cyclase (sAC) and show proliferating neuroprotective astrocytes inhibit microglial activation and downstream neurotoxic astrocyte differentiation to promote retinal ganglion cell (RGC) survival. Finally, we report a new, therapeutically tractable viral vector to specifically target optic nerve head astrocytes and show elevating nuclear or depleting cytoplasmic cAMP in reactive astrocytes inhibits deleterious microglial/macrophage cell activation and promotes RGC survival after optic nerve injury. Thus, soluble adenylyl cyclase and compartmented, nuclear- and cytoplasmic-localized cAMP in reactive astrocytes act as a molecular switch for neuroprotective astrocyte reactivity that can be targeted to inhibit microglial activation and neurotoxic astrocyte differentiation to therapeutic effect. These data expand upon and define new reactive astrocyte subtypes and represent a novel step toward the development of gliotherapeutics for the treatment of glaucoma and other optic neuropathies.

Significance: Retinal neurons are vulnerable to disease and injury, which can result in neuronal death and degeneration leading to irreversible vision loss. The human retina does not regenerate to replace neurons lost to disease or injury. However, cells within the retina of other animals are capable of regenerating neurons, and homologous cells within the mammalian retina could potentially be prompted to do the same. Activating evolutionarily silenced intrinsic regenerative capacity of the mammalian retina could slow, or even reverse, vision loss, leading to an improved quality of life for millions of people. Recent Advances: During development, neurons in the retina are generated progressively by retinal progenitor cells, with distinct neuron types born over developmental time. Many genes function in this process to specify the identity of newly generated neuron types, and these appropriate states of gene expression inform recent regenerative work. When regeneration is initiated in other vertebrates, including birds and fish, specific signaling pathways control the efficiency of regeneration, and these conserved pathways are likely to be important in mammals as well. Critical Issues: Using insights from development and from other animals, limited regeneration from intrinsic cell types has been demonstrated in the mammalian retina, but it is able only to generate a subset of partially differentiated retinal neuron types. Future Directions: Future studies should aim at increasing the efficiency of regeneration, activating regeneration in a targeted fashion across the retina, and improving the ability to generate specific types of retinal neurons to replace those lost to disease or injury. Antioxid. Redox Signal . 39, 1039–1052.

Genetic mosaic analysis, in which mutant cells reside intermingled with wild-type cells, is a powerful experimental approach, but has not been widely used in mice because existing genome-based strategies require complicated and protracted breeding schemes. We have developed an alternative approach termed BEAM (for Binary Expression Aleatory Mosaic) that relies on sparse recombinase activation to generate two genetically distinct, non-overlapping populations of cells for comparative analysis. Following delivery of DNA constructs by transfection or viral transduction, combinatorial recombinase activity generates two distinct populations of cells labeled with either green or red fluorescent protein. Any gene of interest can be mis-expressed or deleted in one population for comparison with intermingled control cells. We have extensively optimized and characterized this system both in vitro and in vivo , and demonstrate its power for investigating cell autonomy, identifying temporally or spatially aberrant phenotypes, revealing changes in cell proliferation or death, and controlling for procedural variability.

Significance: The mammalian central nervous system contains unique projections from the cerebral cortex thought to underpin complex motor and cognitive skills, including the corticospinal tract and corpus callosum. The neurons giving rise to these projections—corticospinal and callosal projection neurons—develop from the same progenitors, but acquire strikingly different fates. The broad evolutionary conservation of known genes controlling cortical projection neuron fates raises the question of how the more narrowly conserved corticospinal and callosal projections evolved. We identify a microRNA cluster selectively expressed by corticospinal projection neurons and exclusive to placental mammals. One of these microRNAs promotes corticospinal fate via regulation of the callosal gene LMO4, suggesting a mechanism whereby microRNA regulation during development promotes evolution of neuronal diversity.

The corticospinal tract is unique to mammals and the corpus callosum is unique to placental mammals (eutherians). The emergence of these structures is thought to underpin the evolutionary acquisition of complex motor and cognitive skills. Corticospinal motor neurons (CSMN) and callosal projection neurons (CPN) are the archetypal projection neurons of the corticospinal tract and corpus callosum, respectively. Although a number of conserved transcriptional regulators of CSMN and CPN development have been identified in vertebrates, none are unique to mammals and most are co-expressed across multiple projection neuron subtypes. Here, we discover seventeen CSMN-enriched microRNAs (miRNAs), fifteen of which map to a single genomic cluster that is exclusive to eutherians. One of these, miR-409-3p, promotes CSMN subtype identity in part via repression of LMO4, a key transcriptional regulator of CPN development. In vivo , miR-409-3p is sufficient to convert deep-layer CPN into CSMN. This is the first demonstration of an evolutionarily acquired miRNA in eutherians that refines cortical projection neuron subtype development. Our findings implicate miRNAs in the eutherians’ increase in neuronal subtype and projection diversity, the anatomic underpinnings of their complex behavior. Significance Statement: The mammalian central nervous system contains unique projections from the cerebral cortex thought to underpin complex motor and cognitive skills, including the corticospinal tract and corpus callosum. The neurons giving rise to these projections - corticospinal and callosal projection neurons - develop from the same progenitors, but acquire strikingly different fates. The broad evolutionary conservation of known genes controlling cortical projection neuron fates raises the question of how the more narrowly conserved corticospinal and callosal projections evolved. We identify a microRNA cluster selectively expressed by corticospinal projection neurons and exclusive to placental mammals. One of these microRNAs promotes corticospinal fate via regulation of the callosal gene LMO4, suggesting a mechanism whereby microRNA regulation during development promotes evolution of neuronal diversity.

Brain mutations, young and old: Most neurons that make up the human brain are postmitotic, living and functioning for a very long time without renewal (see the Perspective by Lee). Baeet al.examined the genomes of single neurons from the prenatal developing human brain. Both the type of mutation and the rates of accumulation changed between gastrulation and neurogenesis. These early mutations could be generating useful neuronal diversity or could predispose individuals to later dysfunction. Lodatoet al.also found that neurons take on somatic mutations as they age by sequencing single neurons from subjects aged 4 months to 82 years. Somatic mutations accumulated with increasing age and accumulated faster in individuals affected by inborn errors in DNA repair. Postmitotic mutations might only affect one neuron, but the accumulated divergence of genomes across the brain could affect function.Science, this issue p.550, p.555; see also p.521

Neuropsychiatric disorders have a complex genetic architecture. Human genetic population-based studies have identified numerous heritable sequence and structural genomic variants associated with susceptibility to neuropsychiatric disease. However, these germline variants do not fully account for disease risk. During brain development, progenitor cells undergo billions of cell divisions to generate the ~80 billion neurons in the brain. The failure to accurately repair DNA damage arising during replication, transcription, and cellular metabolism amid this dramatic cellular expansion can lead to somatic mutations. Somatic mutations that alter subsets of neuronal transcriptomes and proteomes can, in turn, affect cell proliferation and survival and lead to neurodevelopmental disorders. The long life span of individual neurons and the direct relationship between neural circuits and behavior suggest that somatic mutations in small populations of neurons can significantly affect individual neurodevelopment. The Brain Somatic Mosaicism Network has been founded to study somatic mosaicism both in neurotypical human brains and in the context of complex neuropsychiatric disorders.