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Damage to respiratory neural circuitry and consequent loss of diaphragm function is a major cause of morbidity and mortality after cervical spinal cord injury (SCI). Upon SCI, inspiratory signals originating in the medullary rostral ventral respiratory group (rVRG) become disrupted from their phrenic motor neuron (PhMN) targets, resulting in diaphragm paralysis. Limited growth of both damaged and spared axon populations occurs after central nervous system trauma attributed, in part, to expression of various growth inhibitory molecules, some that act through direct interaction with the protein tyrosine phosphatase sigma (PTPσ) receptor located on axons. In the rat model of C2 hemisection SCI, we aimed to block PTPσ signaling to investigate potential mechanisms of axon plasticity and respiratory recovery using a small molecule peptide mimetic that inhibits PTPσ. The peptide was soaked into a biocompatible gelfoam and placed directly over the injury site immediately after hemisection and replaced with a freshly soaked piece 1 week post-SCI. At 8 weeks post-hemisection, PTPσ peptide significantly improved ipsilateral hemidiaphragm function, as assessed in vivo with electromyography recordings. PTPσ peptide did not promote regeneration of axotomized rVRG fibers originating in ipsilateral medulla, as assessed by tracing after adeno-associated virus serotype 2/mCherry injection into the rVRG. Conversely, PTPσ peptide stimulated robust sprouting of contralateral-originating rVRG fibers and serotonergic axons within the PhMN pool ipsilateral to hemisection. Further, relesion through the hemisection did not compromise diaphragm recovery, suggesting that PTPσ peptide-induced restoration of function was attributed to plasticity of spared axon pathways descending in contralateral spinal cord. These data demonstrate that inhibition of PTPσ signaling can promote significant recovery of diaphragm function after SCI by stimulating plasticity of critical axon populations spared by the injury and consequently enhancing descending excitatory input to PhMNs.

Mutations in TUBB4A are associated with a spectrum of neurologic disorders categorized as TUBB4A-related leukoencephalopathy. Affected children can present with global developmental delay or normal early development, followed by a variable loss of skills over time. Further research is needed to characterize the factors associated with the divergent developmental trajectories in this rare monogenic disorder because this phenotypic spectrum is not fully explained by genotype alone. To characterize early psychomotor features, developmental milestones and age of disease onset were collected from medical records (n=54 individuals). Three subcohorts were identified: individuals with the common p.Asp249Asn variant vs all other genotypes with either early ( Our results suggest multiple influences on developmental trajectory, including a strong contribution from genotype and age of onset. Further studies are needed to identify additional factors that influence overall outcomes to better counsel families and to design clinical trials with appropriate clinical endpoints.

More than half of spinal cord injury (SCI) cases occur in the cervical region, leading to respiratory dysfunction due to damaged neural circuitry that controls critically important muscles such as the diaphragm. The C3-C5 spinal cord is the location of phrenic motor neurons (PhMNs) that are responsible for diaphragm activation; PhMNs receive bulbospinal excitatory drive predominately from supraspinal neurons of the rostral ventral respiratory group (rVRG). Cervical SCI results in rVRG axon damage, PhMN denervation, and consequent partial-to-complete paralysis of hemidiaphragm. In a rat model of C2 hemisection SCI, we expressed the axon guidance molecule, brain-derived neurotrophic factor (BDNF), selectively at the location of PhMNs (ipsilateral to lesion) to promote directed growth of rVRG axons toward PhMN targets by performing intraspinal injections of adeno-associated virus serotype 2 (AAV2)-BDNF vector. AAV2-BDNF promoted significant functional diaphragm recovery, as assessed by in vivo electromyography. Within the PhMN pool ipsilateral to injury, AAV2-BDNF robustly increased sprouting of both spared contralateral-originating rVRG axons and serotonergic fibers. Furthermore, AAV2-BDNF significantly increased numbers of putative monosynaptic connections between PhMNs and these sprouting rVRG and serotonergic axons. These findings show that targeting circuit plasticity mechanisms involving the enhancement of synaptic inputs from spared axon populations is a powerful strategy for restoring respiratory function post-SCI.—Charsar, B. A., Brinton, M. A., Locke, K., Chen, A. Y., Ghosh, B., Urban, M. W., Komaravolu, S., Krishnamurthy, K., Smit, R., Pasinelli, P., Wright, M. C., Smith, G. M., Lepore, A. C. AAV2-BDNF promotes respiratory axon plasticity and recovery of diaphragm function following spinal cord injury.

Compromise in inspiratory breathing following cervical spinal cord injury (SCI) is caused by damage to descending bulbospinal axons originating in the rostral ventral respiratory group (rVRG) and consequent denervation and silencing of phrenic motor neurons (PhMNs) that directly control diaphragm activation. In a rat model of high-cervical hemisection SCI, we performed systemic administration of an antagonist peptide directed against phosphatase and tensin homolog (PTEN), a central inhibitor of neuron-intrinsic axon growth potential. PTEN antagonist peptide (PAP4) robustly restored diaphragm function, as determined with electromyography (EMG) recordings in living SCI animals. PAP4 promoted substantial, long-distance regeneration of injured rVRG axons through the lesion and back toward PhMNs located throughout the C3–C5 spinal cord. These regrowing rVRG axons also formed putative excitatory synaptic connections with PhMNs, demonstrating reconnection of rVRG-PhMN-diaphragm circuitry. Lastly, re-lesion through the hemisection site completely ablated functional recovery induced by PAP4. Collectively, our findings demonstrate that axon regeneration in response to systemic PAP4 administration promoted recovery of diaphragmatic respiratory function after cervical SCI.

Stem/progenitor cell transplantation delivery of astrocytes is a potentially powerful strategy for spinal cord injury (SCI). Axon extension into SCI lesions that occur spontaneously or in response to experimental manipulations is often observed along endogenous astrocyte "bridges," suggesting that augmenting this response via astrocyte lineage transplantation can enhance axon regrowth. Given the importance of respiratory dysfunction post-SCI, we transplanted glial-restricted precursors (GRPs)-a class of lineage-restricted astrocyte progenitors-into the C2 hemisection model and evaluated effects on diaphragm function and the growth response of descending rostral ventral respiratory group (rVRG) axons that innervate phrenic motor neurons (PhMNs). GRPs survived long term and efficiently differentiated into astrocytes in injured spinal cord. GRPs promoted significant recovery of diaphragm electromyography amplitudes and stimulated robust regeneration of injured rVRG axons. Although rVRG fibers extended across the lesion, no regrowing axons re-entered caudal spinal cord to reinnervate PhMNs, suggesting that this regeneration response-although impressive-was not responsible for recovery. Within ipsilateral C3-5 ventral horn (PhMN location), GRPs induced substantial sprouting of spared fibers originating in contralateral rVRG and 5-HT axons that are important for regulating PhMN excitability; this sprouting was likely involved in functional effects of GRPs. Finally, GRPs reduced the macrophage response (which plays a key role in inducing axon retraction and limiting regrowth) both within the hemisection and at intact caudal spinal cord surrounding PhMNs. These findings demonstrate that astrocyte progenitor transplantation promotes significant plasticity of rVRG-PhMN circuitry and restoration of diaphragm function and suggest that these effects may be in part through immunomodulation., National Institute of Neurological Disorders and Stroke. Grant Number: 2R01NS079702‐06 (to A.C.L.) Paralyzed Veterans of America Research Foundation. Grant Numbers: Grant 476686 (to A.C.L), 476686 NINDS. Grant Number: 2R01NS079702‐06

Mutations in TUBB4A are associated with a spectrum of neurologic disorders categorized as TUBB4A-related leukoencephalopathy. Affected children can present with global developmental delay or normal early development, followed by a variable loss of skills over time. Further research is needed to characterize the factors associated with the divergent developmental trajectories in this rare monogenic disorder because this phenotypic spectrum is not fully explained by genotype alone. To characterize early psychomotor features, developmental milestones and age of disease onset were collected from medical records (n=54 individuals). Three subcohorts were identified: individuals with the common p.Asp249Asn variant vs all other genotypes with either early ( Our results suggest multiple influences on developmental trajectory, including a strong contribution from genotype and age of onset. Further studies are needed to identify additional factors that influence overall outcomes to better counsel families and to design clinical trials with appropriate clinical endpoints.

Guanylyl cyclase C (GUCY2C) is the afferent central receptor in the gut-brain endocrine axis regulated by the anorexigenic intestinal hormone uroguanylin. GUCY2C mRNA and protein are produced in the hypothalamus, a major center regulating appetite and metabolic homeostasis. Further, GUCY2C mRNA and protein are expressed in the ventral midbrain, a principal structure regulating hedonic reward from behaviors including eating. While GUCY2C is expressed in hypothalamus and midbrain, its precise neuroanatomical organization and relationship with circuits regulating satiety remain unknown. Here, we reveal that hypothalamic GUCY2C mRNA is confined to the ventral premammillary nucleus (PMV), while in midbrain it is produced by neurons in the ventral tegmental area (VTA) and substantia nigra (SN). GUCY2C in the PMV is produced by 46% of neurons expressing anorexigenic leptin receptors, while in the VTA/SN it is produced in most tyrosine hydroxylase-immunoreactive neurons. In contrast to mRNA, GUCY2C protein is widely distributed throughout the brain in canonical sites of PMV and VTA/SN axonal projections. Selective stereotaxic ablation of PMV or VTA/SN neurons eliminated GUCY2C only in their respective canonical projection sites. Conversely, specific anterograde tracer analyses of PMV or VTA/SN neurons confirmed distinct GUCY2C-immunoreactive axons projecting to those canonical locations. Together, these findings reveal two discrete neuronal circuits expressing GUCY2C originating in the PMV in the hypothalamus and in the VTA/SN in midbrain, which separately project to other sites throughout the brain. They suggest a structural basis for a role for the GUCY2C-uroguanylin gut-brain endocrine axis in regulating homeostatic and behavioral components contributing to satiety.

The therapeutic benefit of cell transplantation has been assessed in a host of central nervous system (CNS) diseases, including disorders of the spinal cord such as traumatic spinal cord injury (SCI). The promise of cell transplantation to preserve and/or restore normal function can be aimed at a variety of therapeutic mechanisms, including replacement of lost or damaged CNS cell types, promotion of axonal regeneration or sprouting, neuroprotection, immune response modulation, and delivery of gene products such as neurotrophic factors, amongst other possibilities. Despite significant work in the field of transplantation in models of SCI, limited attention has been directed at harnessing the therapeutic potential of cell grafting for preserving respiratory function after SCI, despite the critical role pulmonary compromise plays in patient outcome in this devastating disease. Here, we will review the limited number of studies that have demonstrated the therapeutic potential of intraspinal transplantation of a variety of cell types for addressing respiratory dysfunction in SCI.

Knobloch and Layer described a triad of symptoms – vitreoretinal degeneration, high myopia, and congenital encephalocele – in 5 affected siblings in a large consanguineous family.1 The cause of what is now Knobloch syndrome (KS) was identified as mutation of COL18A1, the gene encoding collagen XVIII.2 We present an unusual case of two brothers with severe global developmental delay, medically intractable epilepsy, diffuse polymicrogyria, and high myopia, born to non-consanguineous parents of Palestinian ethnicity. Although the brothers lack typically-associated skull defects, they were found to harbor a novel homozygous pathogenic mutation in COL18A1, leading to a diagnosis of KS on the severe end of the phenotypic spectrum. Epilepsy and brain malformations are uncommon in cases of KS; hence, this report expands the current clinical spectrum and reinforces the diagnostic utility of trio whole exome sequencing analysis in appropriately selected cases of polymicrogyria.

Toxoplasma gondii infection occurs through the oral route, but we lack important information about how the parasite interacts with the host immune system in the intestine. We used two-photon laser-scanning microscopy in conjunction with a mouse model of oral T. gondii infection to address this issue. T. gondii established discrete foci of infection in the small intestine, eliciting the recruitment and transepithelial migration of neutrophils and inflammatory monocytes. Neutrophils accounted for a high proportion of actively invaded cells, and we provide evidence for a role for transmigrating neutrophils and other immune cells in the spread of T. gondii infection through the lumen of the intestine. Our data identify neutrophils as motile reservoirs of T. gondii infection and suggest a surprising retrograde pathway for parasite spread in the intestine.

The highly conserved FACT (Facilitates Chromatin Transactions) complex performs essential functions in eukaryotic cells through the reorganization of nucleosomes. During transcription, FACT reorganizes nucleosomes to allow passage of RNA Polymerase II and then assists in restoring these nucleosomes after RNA Polymerase II has passed. We have previously shown, consistent with this function, that Spt16 facilitates repression of the Saccharomyces cerevisiae SER3 gene by maintaining nucleosome occupancy over the promoter of this gene as a consequence of intergenic transcription of SRG1 noncoding DNA. In this study, we report the results of a genetic screen to identify mutations in SPT16 that derepress SER3. Twenty-five spt16 mutant alleles were found to derepress SER3 without causing significant reductions in either SRG1 RNA levels or Spt16 protein levels. Additional phenotypic assays indicate that these mutants have general transcription defects related to altered chromatin structure. Our analyses of a subset of these spt16 mutants reveal defects in SRG1 transcription-coupled nucleosome occupancy over the SER3 promoter. We provide evidence that these mutants broadly impair transcription-coupled nucleosome occupancy at highly transcribed genes but not at lowly transcribed genes. Finally, we show that one consequence shared by these mutations is the reduced binding of mutant Spt16 proteins across SRG1 and other highly transcribed genes. Taken together, our results highlight an important role for Spt16 in orchestrating transcription-coupled nucleosome assembly at highly transcribed regions of the genome, possibly by facilitating the association of Spt16 during this process.