Biallelic CRELD1 variants cause a multisystem syndrome, including neurodevelopmental phenotypes, cardiac dysrhythmias, and frequent infections.

PURPOSE: We sought to delineate a multisystem disorder caused by recessive cysteine-rich with epidermal growth factor-like domains 1 (CRELD1) gene variants. METHODS: The impact of CRELD1 variants was characterized through an international collaboration utilizing next-generation DNA sequencing, gene knockdown, and protein overexpression in Xenopus tropicalis, and in vitro analysis of patient immune cells. RESULTS: Biallelic variants in CRELD1 were found in 18 participants from 14 families. Affected individuals displayed an array of phenotypes involving developmental delay, early-onset epilepsy, and hypotonia, with about half demonstrating cardiac arrhythmias and some experiencing recurrent infections. Most harbored a frameshift in trans with a missense allele, with 1 recurrent variant, p.(Cys192Tyr), identified in 10 families. X tropicalis tadpoles with creld1 knockdown displayed developmental defects along with increased susceptibility to induced seizures compared with controls. Additionally, human CRELD1 harboring missense variants from affected individuals had reduced protein function, indicated by a diminished ability to induce craniofacial defects when overexpressed in X tropicalis. Finally, baseline analyses of peripheral blood mononuclear cells showed similar proportions of immune cell subtypes in patients compared with healthy donors. CONCLUSION: This patient cohort, combined with experimental data, provide evidence of a multisystem clinical syndrome mediated by recessive variants in CRELD1.

Fetal Brain-Derived Exosomal miRNAs from Maternal Blood: Potential Diagnostic Biomarkers for Fetal Alcohol Spectrum Disorders (FASDs).

Fetal alcohol spectrum disorders (FASDs) are leading causes of neurodevelopmental disability but cannot be diagnosed early in utero. Because several microRNAs (miRNAs) are implicated in other neurological and neurodevelopmental disorders, the effects of EtOH exposure on the expression of these miRNAs and their target genes and pathways were assessed. In women who drank alcohol (EtOH) during pregnancy and non-drinking controls, matched individually for fetal sex and gestational age, the levels of miRNAs in fetal brain-derived exosomes (FB-Es) isolated from the mothers' serum correlated well with the contents of the corresponding fetal brain tissues obtained after voluntary pregnancy termination. In six EtOH-exposed cases and six matched controls, the levels of fetal brain and maternal serum miRNAs were quantified on the array by qRT-PCR. In FB-Es from 10 EtOH-exposed cases and 10 controls, selected miRNAs were quantified by ddPCR. Protein levels were quantified by ELISA. There were significant EtOH-associated reductions in the expression of several miRNAs, including miR-9 and its downstream neuronal targets BDNF, REST, Synapsin, and Sonic hedgehog. In 20 paired cases, reductions in FB-E miR-9 levels correlated strongly with reductions in fetal eye diameter, a prominent feature of FASDs. Thus, FB-E miR-9 levels might serve as a biomarker to predict FASDs in at-risk fetuses.

Spectrum of qualitative and quantitative imaging of pilomyxoid, intermediate pilomyxoid and pilocytic astrocytomas in relation to their genetic alterations.

PURPOSE: Pilomyxoid astrocytomas (PMA) are pediatric brain tumors predominantly located in the suprasellar region, third ventricle and posterior fossa, which are considered to be more clinically aggressive than pilocytic astrocytomas (PA). Another entity, intermediate pilomyxoid tumors (IPT), exists within the spectrum of pilocytic/pilomyxoid astrocytomas. The 2021 WHO CNS classification refrained from assigning grade 1 or 2 status to PMA, thereby reflecting the need to further elucidate their clinical and imaging characteristics. METHODS: We included a total of 15 patients with PMA, IPT and suprasellar PA. We retrospectively evaluated immunohistochemistry, imaging findings and diffusion characteristics within these tumors as well as whole exome sequencing for three of the cases. RESULTS: 87% of the tumors were supratentorial with 11 cases suprasellar in location, 1 case located in the frontal white matter and 1 in the hippocampus. 6 cases demonstrated intraventricular extension. ADC values were higher in PMA and IPT than PA. 3 cases demonstrated KIAA1549-BRAF-fusion, 2 had BRAF[Formula: see text]-mutation and 6 were BRAF-wildtype. All cases had recurrence/progression on follow-up. CONCLUSION: PMA and IPT do not demonstrate aggressive imaging characteristics in respect to their diffusion imaging with ADC values being higher than PA. Lack of BRAF-alteration in PMA corresponded to atypical location of tumors with atypical driver mutations and mechanisms.

Effects of In Utero EtOH Exposure on 18S Ribosomal RNA Processing: Contribution to Fetal Alcohol Spectrum Disorder.

Fetal alcohol spectrum disorders (FASD) are leading causes of neurodevelopmental disability. The mechanisms by which alcohol (EtOH) disrupts fetal brain development are incompletely understood, as are the genetic factors that modify individual vulnerability. Because the phenotype abnormalities of FASD are so varied and widespread, we investigated whether fetal exposure to EtOH disrupts ribosome biogenesis and the processing of pre-ribosomal RNAs and ribosome assembly, by determining the effect of exposure to EtOH on the developmental expression of 18S rRNA and its cleaved forms, members of a novel class of short non-coding RNAs (srRNAs). In vitro neuronal cultures and fetal brains (11-22 weeks) were collected according to an IRB-approved protocol. Twenty EtOH-exposed brains from the first and second trimester were compared with ten unexposed controls matched for gestational age and fetal gender. Twenty fetal-brain-derived exosomes (FB-Es) were isolated from matching maternal blood. RNA was isolated using Qiagen RNA isolation kits. Fetal brain srRNA expression was quantified by ddPCR. srRNAs were expressed in the human brain and FB-Es during fetal development. EtOH exposure slightly decreased srRNA expression (1.1-fold; p = 0.03). Addition of srRNAs to in vitro neuronal cultures inhibited EtOH-induced caspase-3 activation (1.6-fold, p = 0.002) and increased cell survival (4.7%, p = 0.034). The addition of exogenous srRNAs reversed the EtOH-mediated downregulation of srRNAs (2-fold, p = 0.002). EtOH exposure suppressed expression of srRNAs in the developing brain, increased activity of caspase-3, and inhibited neuronal survival. Exogenous srRNAs reversed this effect, possibly by stabilizing endogenous srRNAs, or by increasing the association of cellular proteins with srRNAs, modifying gene transcription. Finally, the reduction in 18S rRNA levels correlated closely with the reduction in fetal eye diameter, an anatomical hallmark of FASD. The findings suggest a potential mechanism for EtOH-mediated neurotoxicity via alterations in 18S rRNA processing and the use of FB-Es for early diagnosis of FASD. Ribosome biogenesis may be a novel target to ameliorate FASD in utero or after birth. These findings are consistent with observations that gene-environment interactions contribute to FASD vulnerability.

Molecular Markers in Maternal Blood Exosomes Allow Early Detection of Fetal Alcohol Spectrum Disorders.

Prenatal alcohol exposure can cause developmental abnormalities (fetal alcohol spectrum disorders; FASD), including small eyes, face and brain, and neurobehavioral deficits. These cannot be detected early in pregnancy with available imaging techniques. Early diagnosis could facilitate development of therapeutic interventions. Banked human fetal brains and eyes at 9−22 weeks' gestation were paired with maternal blood samples, analyzed for morphometry, protein, and RNA expression, and apoptotic signaling. Alcohol (EtOH)-exposed (maternal self-report) fetuses were compared with unexposed controls matched for fetal age, sex, and maternal race. Fetal brain-derived exosomes (FB-E) were isolated from maternal blood and analyzed for protein, RNA, and apoptotic markers. EtOH use by mothers, assessed by self-report, was associated with reduced fetal eye diameter, brain size, and markers of synaptogenesis. Brain caspase-3 activity was increased. The reduction in eye and brain sizes were highly correlated with amount of EtOH intake and caspase-3 activity. Levels of several biomarkers in FB-E, most strikingly myelin basic protein (MBP; r > 0.9), correlated highly with morphological abnormalities. Reduction in FB-E MBP levels was highly correlated with EtOH exposure (p < 1.0 × 10−10). Although the morphological features of FAS appear long before they can be detected by live imaging, FB-E in the mother's blood may contain markers, particularly MBP, that predict FASD.

Ethanol-mediated alterations in oligodendrocyte differentiation in the developing brain.

INTRODUCTION: Alterations of white matter integrity and subsequent white matter structural deficits are consistent findings in Fetal Alcohol Syndrome (FAS), but knowledge regarding the molecular mechanisms underlying these abnormalities is incomplete. Experimental rodent models of FAS have shown dysregulation of cytokine expression leading to apoptosis of oligodendrocyte precursor cells (OPCs) and altered oligodendrocyte (OL) differentiation, but whether this is representative of human FAS pathogenesis has not been determined. METHODS: Fetal brain tissue (12.2-21.4 weeks gestation) from subjects undergoing elective termination of pregnancy was collected according to an IRB-approved protocol. Ethanol (EtOH) exposure status was classified based on a detailed face-to-face questionnaire adapted from the National Institute on Alcohol Abuse and Alcoholism Prenatal Alcohol and Sudden Infant Death Syndrome and Stillbirth (PASS) study. Twenty EtOH-exposed fetuses were compared with 20 gestational age matched controls. Cytokine and OPC marker mRNA expression was quantified by Real-Time Polymerase chain reaction (qRT-PCR). Patterns of protein expression of OPC markers and active Capase-3 were studied by Fluorescence Activated Cell Sorting (FACS). RESULTS: EtOH exposure was associated with reduced markers of cell viability, OPC differentiation, and OL maturation, while early OL differentiation markers were unchanged or increased. Expression of mRNAs for proteins specific to more mature forms of OL lineage (platelet-derived growth factor α (PDGFRα) and myelin basic protein (MBP) was lower in the EtOH group than in controls. Expression of the multifunctional growth and differentiation-promoting growth factor IGF-1, which is essential for normal development, also was reduced. Reductions were not observed for markers of early stages of OL differentiation, including Nuclear transcription factor NK-2 homeobox locus 2 (Nkx2.2). Expression of mRNAs for the proinflammatory cytokine, tumor necrosis factor-α (TNFα), and several proinflammatory chemokines was higher in the EtOH group compared to controls, including: Growth regulated protein alpha/chemokine (C-X-C motif) ligand 1 (GRO-α/CXCL1), Interleukin 8/chemokine (C-X-C motif) ligand 8 (IL8/CXCL8), Chemokine (C-X-C motif) ligand 6/Granulocyte chemotactic protein 2 (CXCL16/GCP2), epithelial-derived neutrophil-activating protein 78/chemokine (C-X-C motif) ligand 5 (ENA-78/CXCL5), monocyte chemoattractant protein-1 (MCP-1). EtOH exposure also was associated with an increase in the proportion of cells expressing markers of early stage OPCs, such as A2B5 and NG2. Finally, apoptosis (measured by caspase-3 activation) was increased substantially in the EtOH group compared to controls. CONCLUSION: Prenatal EtOH exposure is associated with excessive OL apoptosis and/or delayed OL maturation in human fetal brain. This is accompanied by markedly dysregulated expression of several chemokines and cytokines, in a pattern predictive of increased OL cytotoxicity and reduced OL differentiation. These findings are consistent with findings in animal models of FAS.

Unilateral leg pain caused by cryptococcal myositis: An unusual presentation of disseminated cryptococcosis in a kidney transplant recipient.

Cryptococcal disease is a rare but often serious infection in solid organ transplant recipients, commonly presenting as meningitis and pneumonia but can rarely cause myositis. We report the case of a 43-year-old female kidney transplant recipient with two previous graft failures requiring re-transplantations who presented with a 1-month duration of worsening unilateral leg pain, swelling, and shortness of breath. Blood cultures isolated Cryptococcus neoformans. A calf biopsy was performed and histopathology revealed myonecrosis with yeast forms consistent with Cryptococcus spp. Liposomal amphotericin B (LamB) was administered. Her course was complicated by hypoxemic respiratory failure with development of ground glass opacities on chest imaging. Work-up revealed bacterial and C neoformans pneumonia and probable Pneumocystis jirovecii pneumonia (PJP) She received trimethoprim-sulfamethoxazole and LamB and was discharged on fluconazole. Shortly thereafter she was re-admitted with confusion, septic shock, and multi-organ failure. Work-up revealed PJP with subsequent development of cryptococcal meningitis. Despite aggressive management, she expired. Disseminated cryptococcal infection may manifest as myositis. Presence of cryptococcal infection is a marker of severe net state of immunosuppression (IS), hence, presence of other opportunistic infections is likely. Early recognition of cryptococcal infection, institution of targeted therapy, and IS reduction are important to improve overall survival.

TNF-α/TNFR2 Regulatory Axis Stimulates EphB2-Mediated Neuroregeneration Via Activation of NF-κB.

HIV-1 infected individuals are at high risk of developing HIV-associated neurocognitive disorders (HAND) as HIV infection leads to neuronal injury and synaptic loss in the central nervous system (CNS). The neurotoxic effects of HIV-1 are primarily a result of viral replication leading to the production of inflammatory chemokines and cytokines, including TNF-α. Given an important role of TNF-α in regulating synaptic plasticity, we investigated the effects of TNF-α on the development of neuronal processes after mechanical injury, and we showed that TNF-α treatment stimulates the regrowth of neuronal processes. To investigate transcriptional effects of TNF-α on synaptic plasticity, we analyzed both human neurosphere and isolated neuronal cultures for the regulation of genes central to synaptic alterations during learning and memory. TNF-α treatment upregulated Ephrin receptor B2 (EphB2), which is strongly involved in dendritic arborization and synaptic integrity. TNF-α strongly activates the NF-κB pathway, therefore, we propose that TNF-α-induced neurite regrowth occurs primarily through EphB2 signaling via stimulation of NF-κB. EphB2 promoter activity increased with TNF-α treatment and overexpression of NF-κB. Direct binding of NF-κB to the EphB2 promoter occurred in the ChIP assay, and site-directed mutagenesis identified binding sites involved in TNF-α-induced EphB2 activation. TNF-α induction of EphB2 was determined to occur specifically through TNF-α receptor 2 (TNFR2) activation in human primary fetal neurons. Our observations provide a new avenue for the investigation on the impact of TNF-α in the context of HIV-1 neuronal cell damage as well as providing a potential therapeutic target in TNFR2 activation of EphB2.

Involvement of IRS-1 interaction with ADAM10 in the regulation of neurite extension.

The insulin-like growth factor-1 (IGF-1) signaling pathway plays an important role in neuronal cell differentiation. Recent studies have shown that IGF-1 has the capacity to counteract the retraction of neuronal processes in response to inflammatory cytokines such as TNF-α, which is a known factor for neuronal injury in the central nervous system. This event is thought to be mediated via interference of TNF-α-induced interaction of ß1-integrin with insulin receptor substrate-1 (IRS-1). Here, we demonstrate the interaction of IRS-1 with disintegrin and metalloproteinase ADAM10 through the N-terminal domain of IRS-1 and that this is involved in the regulation of neurite extension and retraction by IGF-1 and TNF-α, respectively. PC12 cells expressing the N-terminal domain show enhanced neurite extension after IGF-1 treatment and reduced neurite depletion relative to control cells after TNF-α treatment. The level of ADAM10 was found to be increased in immunohistochemical studies of HIV encephalitis clinical samples and is present with TNF-α and TNFR1 in both astrocytes and neurons. Altogether, these observations suggest a role for ADAM10 in the mechanism for IGF1/IRS-1 signaling pathway in sustaining the stability of neuronal processes.

Molecular Genetics Augment Cytopathologic Evaluation and Surgical Planning of Pediatric Thyroid Nodules.

PURPOSE: Molecular genetic testing in conjunction with cytopathology may improve prediction of malignancy in thyroid nodules, particularly those with indeterminate cytology (Bethesda III/IV). Though now commonplace in adults, pediatric data are limited. This study examines molecular genetics of pediatric nodules with correlation to cytologic and histologic classification at time of surgery and the distribution of mutations. METHODS: Retrospective chart review of 164 patients <22 years who underwent surgical resection of a thyroid nodule between 2002 and 2020 with molecular testing on fine-needle aspiration biopsy (FNA) or final histopathology. RESULTS: 85 (52 %) of 164 patients undergoing thyroid resection had available molecular genetic testing. BRAF V600E testing was performed on the FNA samples of 73 (86 %) patients and on 15 (18 %) surgical specimens; 31 (37 %) were positive. Of the remaining 54 patients, 21 had additional mutation/fusion testing. In 17 (81 %) cases, an alternate mutation/fusion was identified including 8 gene fusions, 3 DICER1 mutations, 4 NRAS mutations, one BRAF variant, and one unknown variant. BRAF, DICER1 mutations, and gene fusions predicted malignancy. Greater than 95 % of BRAF mutations were in Bethesda V/VI lesions and associated with classic variant PTC whereas fusions and DICER1 mutations clustered in Bethesda IV nodules. Bethesda III nodules harbored BRAF and NRAS mutations. In Bethesda IV nodules, a gene fusion or DICER mutation altered the surgical decision-making (upfront thyroidectomy rather than lobectomy) in 70 % of nodules submitted for genetic testing. CONCLUSION: Expanded molecular genetic testing on FNA of pediatric thyroid nodules, particularly Bethesda III/IV, may improve prediction of malignancy and augment surgical decision-making. LEVEL OF EVIDENCE: III.

Polyomavirus JC infection inhibits differentiation of oligodendrocyte progenitor cells.

Reactivation of the human polyomavirus JC (JCV) in the CNS results in a fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). The lytic destruction of oligodendrocytes, which occurs at the terminal stage of the viral infection cycle, is considered a critical factor in the development of demyelination and the pathogenesis of PML. However, knowledge is limited about interaction of JCV with oligodendrocytes and its impact on the denudation of axons at the early stage of viral reactivation and prior to the destruction of the infected cells. We have developed an in vitro neuroprogenitor cell culture using human fetal brain that can be differentiated to the oligodendrocyte lineage to investigate interactions of JCV with its host cells. Results show that infection with JCV delays oligodendrocyte maturation as shown by reduced levels of oligodendrocytic markers, including myelin basic protein, proteolipid protein, and platelet-derived growth factor receptor-α. Furthermore, replication of JCV in these cells caused substantial dysregulation of several chemokines, including CCL5/RANTES, GRO, CXCL1/GROα, CXCL16, CXCL8/IL-8, CXCL5/ENA-78, and CXCL10/IP-10, all of which play a role in cell growth and differentiation.

In utero ethanol exposure induces mitochondrial DNA damage and inhibits mtDNA repair in developing brain.

Introduction: Mitochondrial dysfunction is postulated to be a central event in fetal alcohol spectrum disorders (FASD). People with the most severe form of FASD, fetal alcohol syndrome (FAS) are estimated to live only 34 years (95% confidence interval, 31 to 37 years), and adults who were born with any form of FASD often develop early aging. Mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage, hallmarks of aging, are postulated central events in FASD. Ethanol (EtOH) can cause mtDNA damage, consequent increased oxidative stress, and changes in the mtDNA repair protein 8-oxoguanine DNA glycosylase-1 (OGG1). Studies of molecular mechanisms are limited by the absence of suitable human models and non-invasive tools. Methods: We compared human and rat EtOH-exposed fetal brain tissues and neuronal cultures, and fetal brain-derived exosomes (FB-Es) from maternal blood. Rat FASD was induced by administering a 6.7% alcohol liquid diet to pregnant dams. Human fetal (11-21 weeks) brain tissue was collected and characterized by maternal self-reported EtOH use. mtDNA was amplified by qPCR. OGG1 and Insulin-like growth factor 1 (IGF-1) mRNAs were assayed by qRT-PCR. Exosomal OGG1 was measured by ddPCR. Results: Maternal EtOH exposure increased mtDNA damage in fetal brain tissue and FB-Es. The damaged mtDNA in FB-Es correlated highly with small eye diameter, an anatomical hallmark of FASD. OGG1-mediated mtDNA repair was inhibited in EtOH-exposed fetal brain tissues. IGF-1 rescued neurons from EtOH-mediated mtDNA damage and OGG1 inhibition. Conclusion: The correlation between mtDNA damage and small eye size suggests that the amount of damaged mtDNA in FB-E may serve as a marker to predict which at risk fetuses will be born with FASD. Moreover, IGF-1 might reduce EtOH-caused mtDNA damage and neuronal apoptosis.

Biomarkers of Affective Dysregulation Associated with In Utero Exposure to EtOH.

INTRODUCTION: Children with fetal alcohol spectrum disorders (FASD) exhibit behavioral and affective dysregulation, including hyperactivity and depression. The mechanisms are not known, but they could conceivably be due to postnatal social or environmental factors. However, we postulate that, more likely, the affective dysregulation is associated with the effects of EtOH exposure on the development of fetal serotonergic (5-HT) and/or dopaminergic (DA) pathways, i.e., pathways that in postnatal life are believed to regulate mood. Many women who use alcohol (ethanol, EtOH) during pregnancy suffer from depression and take selective serotonin reuptake inhibitors (SSRIs), which might influence these monoaminergic pathways in the fetus. Alternatively, monoaminergic pathway abnormalities might reflect a direct effect of EtOH on the fetal brain. To distinguish between these possibilities, we measured their expressions in fetal brains and in fetal brain-derived exosomes (FB-Es) isolated from the mothers' blood. We hypothesized that maternal use of EtOH and/or SSRIs during pregnancy would be associated with impaired fetal neural development, detectable as abnormal levels of monoaminergic and apoptotic biomarkers in FB-Es. METHODS: Fetal brain tissues and maternal blood were collected at 9-23 weeks of pregnancy. EtOH groups were compared with unexposed controls matched for gestational age (GA). The expression of 84 genes associated with the DA and 5-HT pathways was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) on microarrays. FB-Es also were assayed for serotonin transporter protein (SERT) and brain-derived neurotrophic factor (BDNF) by enzyme-linked immunosorbent assay (ELISA). RESULTS: Six EtOH-exposed human fetal brain samples were compared to SSRI- or polydrug-exposed samples and to unexposed controls. EtOH exposure was associated with significant upregulation of DA receptor D3 and 5-HT receptor HTR2C, while HTR3A was downregulated. Monoamine oxidase A (MAOA), MAOB, the serine/threonine kinase AKT3, and caspase-3 were upregulated, while mitogen-activated protein kinase 1 (MAPK1) and AKT2 were downregulated. ETOH was associated with significant upregulation of the DA transporter gene, while SERT was downregulated. There were significant correlations between EtOH exposure and (a) caspase-3 activation, (b) reduced SERT protein levels, and (c) reduced BDNF levels. SSRI exposure independently increased caspase-3 activity and downregulated SERT and BDNF. Early exposure to EtOH and SSRI together was associated synergistically with a significant upregulation of caspase-3 and a significant downregulation of SERT and BDNF. Reduced SERT and BDNF levels were strongly correlated with a reduction in eye diameter, a somatic manifestation of FASD. CONCLUSIONS: Maternal use of EtOH and SSRI during pregnancy each was associated with changes in fetal brain monoamine pathways, consistent with potential mechanisms for the affective dysregulation associated with FASD.

Maternal Blood Lipid Biomarkers of Oligodendrocyte Pathology to Predict Fetal Alcohol Spectrum Disorders.

Introduction: Up to 9.9% of children have fetal alcohol spectrum disorders (FASD), the most frequent cause of intellectual disability in the US. FASD may involve abnormal brain development, including dysmyelination, suggesting abnormal development of oligodendrocytes (OLs), which make myelin and are rich in lipids. Indeed, low serum levels of omega-3 fatty acids (ω-3) have been reported in FASD. Free fatty acids bind to specific receptors (FFARs). We have isolated cell type-specific fetal brain-derived exosomes (FB-E) from maternal blood and sampled their contents to search for lipid-related biomarkers that predict FASD. Methods: Blood samples were collected from two groups of pregnant women: 1) those who consumed EtOH during pregnancy, and 2) non-EtOH using controls, under an IRB-approved protocol. Serum and OL-derived exosomes (OL-Es) were used to assay myelin basic protein (MBP) and FFAR by ELISA and droplet digital PCR (ddPCR), respectively. Results: FFAR and MBP proteins were downregulated in the EtOH group compared to controls, and this difference was greatest in OL-Es from maternal blood compared maternal serum. Conclusion: MBP and FFAR levels were reduced in OL-Es from EtOH-consuming pregnant women. The data suggest potential therapeutic targets to predict which children are at risk for developing FASD and reduce dysmyelination in developing.

Exosomal Lipid Biomarkers of Oligodendrocyte Pathology to Predict Scoliosis in Children with Cerebral Palsy.

Introduction: Cerebral Palsy (CP), the most common cause of disability in children, is phenotypically heterogeneous. Approximately 20% of cases develop severe scoliosis. A pathological hallmark of CP is periventricular leukomalacia (PVL), which is due to dysmyelination, suggesting the possibility of a lipidomic abnormality. Risk factors for CP include perinatal hypoxia, prematurity, multiple gestation, ischemia, infection, and maternal alcohol consumption. There is evidence for low serum levels of omega-3 (ω-3) fatty acids in CP patients, and separately in idiopathic scoliosis. Many effects of free fatty acids (FFAs) are mediated via specific G protein-coupled free fatty acid receptors (FFARs), which play essential roles as nutritional and signaling molecules. FFAs, including ω-3, and their receptors are involved in the development and metabolism of oligodendrocytes (OLs), and are critical to myelination. Thus, the cases of CP that will develop severe scoliosis might be those in which there is a deficiency of ω-3, FFARs, or other lipidomic abnormality that is detectable early in the plasma. If so, we might be able to predict scoliosis and prevent it with dietary supplementation. Methods: Blood samples were collected from four groups of patients at the Philadelphia Shriners Children's Hospital (SCH-P): 1) patients with CP; 2) severe scoliosis (>40o); 3) CP plus scoliosis; and 4) non-impaired controls stratified by age (2-18 yrs), gender, and race/ethnicity, under an IRB-approved protocol. Serum proteins and RNA were purified, and OL-derived exosomes (OL-Es) isolated, using myelin basic protein (MBP) as a late OL marker. Protein was used for the detection of MBP and FFAR by enzyme-linked immunosorbent assays (ELISAs), and by flow cytometry. RNA was assayed by digital droplet polymerase chain reaction (ddPCR) for OL markers and FFAR expression. Results: FFAR and MBP proteins were downregulated in each of the three patient groups compared to controls, and this difference was greatest in both patients with CP plus scoliosis. Conclusion: Altogether, MBP and FFAR levels were reduced in OL-Es from both children with CP plus scoliosis. The lipid abnormalities specific to CP with scoliosis were concentrated in OLs. Our data might i) suggest therapeutic targets to reduce dysmyelination and scoliosis in CP, ii) predict which children are at risk for developing scoliosis, iii) lead to therapeutic trials of fatty acids for CP and other dysmyelinating neurological disorders.

JCV agnoprotein-induced reduction in CXCL5/LIX secretion by oligodendrocytes is associated with activation of apoptotic signaling in neurons.

An indispensable role for oligodendrocytes in the protection of axon function and promotion of neuronal survival is strongly supported by the finding of progressive neuron/axon degeneration in human neurological diseases that affect oligodendrocytes. Imaging and pathological studies of the CNS have shown the presence of neuroaxonal injury in progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the CNS, resulting from destruction of oligodendrocytes upon productive replication of the pathogenic neurotropic polyomavirus JC. Here, we examined the extracellular factors involved in communication between oligodendrocytes and neurons. Culturing cortical neurons with conditioned medium (CM) from rat CG4 oligodendrocytic cells that express the JCV agnoprotein showed that CXCL5/LIX, which is a chemokine closely related to the human CXCL5/ENA78 and CXCL6/GCP-2 chemokines, is essential for neuronal cell survival. We found that in CM from agnoprotein-producing CG-4 cells level of CXC5/LIX is decreased compared to control cells. We also demonstrated that a reduced expression of CXCL5/LIX by CG4 GFP-Agno cells triggered a cascade of signaling events in cortical neurons. Analysis of mitogen-activated protein kinases (MAPK) and glycogen synthase kinase (GSK3) pathways showed that they are involved in mechanisms of neuronal apoptosis in response to the depletion of CXCL5/LIX signaling. These data suggest that agnoprotein-induced dysregulation of chemokine production by oligodendrocytes may contribute to neuronal/axonal injury in the pathogenesis of PML lesions.

Histological changes associated with laser interstitial thermal therapy for radiation necrosis: illustrative cases.

BACKGROUND: Patients with lung cancer and melanoma remain the two largest groups to develop brain metastases. Immunotherapy has been approved for treatment of stage IV disease in both groups. Many of these patients are additionally treated with stereotactic radiosurgery for their brain metastases during ongoing immunotherapy. Use of immunotherapy has been reported to increase the rates of radiation necrosis (RN) after radiosurgery, causing neurological compromise due to growth of the enhancing lesion as well as worsening of associated cerebral edema. OBSERVATIONS: Laser interstitial thermal therapy (LITT) is a surgical approach that has been shown effective in the management of RN, especially given its efficacy in early reduction of perilesional edema. However, little remains known about the pathology of the post-LITT lesions and how LITT works in this condition. Here, we present two patients who needed surgical decompression after LITT for RN. Clinical, histopathological, and imaging features of both patients are presented. LESSONS: Criteria for selecting the best patients with RN for LITT therapy remains unclear. Given two similarly sized lesions and not too dissimilar clinical histories but with differing outcomes, further investigation is clearly needed to identify predictors of response to LITT in the setting of SRS and immunotherapy-induced RN.

Biallelic inactivation of PBRM1 as a molecular driver in a rare pineoblastoma case: illustrative case.

BACKGROUND: Pineoblastomas are a rare and aggressive pediatric neuroectodermal tumor subtype. Because of their rarity, pineoblastomas are still poorly understood, and there is little research delineating their molecular development and underlying genetic phenotype. Recent multiomic studies in pineoblastomas and pineal parenchymal tumors identified four clinically and biologically relevant consensus groups driven by signaling/processing pathways; however, molecular level alterations leading to these pathway changes are yet to be discovered, hence the importance of individually profiling every case of this rare tumor type. OBSERVATIONS: The authors present the comprehensive somatic genomic profiling of a patient with pineoblastoma presenting with the loss of protein polybromo-1 (PBRM1) as a candidate genomic driver. Loss of PBRM1, a tumor suppressor, has been reported as a driver event in various cancer types, including renal cell carcinoma, bladder carcinoma, and meningiomas with papillary features. LESSONS: This is the first report presenting biallelic loss of PBRM1 as a candidate molecular driver in relation to pineoblastoma.

Characterization of perivascular space pathology in a rat model of cerebral small vessel disease by in vivo magnetic resonance imaging.

One of the most common causes of dementia is cerebral small vessel disease (SVD), which is associated with enlarged perivascular spaces (PVS). Clinically, PVS are visible as hyperintensities on T2-weighted (T2w) magnetic resonance images (MRI). While rodent SVD models exhibit arteriolosclerosis, PVS have not been robustly documented by MRI casting doubts on their clinical relevance. Here we established that the severity of SVD in spontaneously hypertensive stroke prone (SHRSP) rats correlated to 'moderate' SVD in human post-mortem tissue. We then developed two approaches for detecting PVS in SHRSP rats: 1) T2w imaging and 2) T1-weighted imaging with administration of gadoteric acid into cerebrospinal fluid. We applied the two protocols to six Wistar-Kyoto (WKY) control rats and thirteen SHRSP rats at ∼12 month of age. The primary endpoint was the number of hyperintense lesions. We found more hyperintensities on T2w MRI in the SHRSP compared to WKY rats (p-value = 0.023). CSF enhancement with gadoteric acid increased the visibility of PVS-like lesions in SHRSP rats. In some of the SHRSP rats, the MRI hyperintensities corresponded to enlarged PVS on histopathology. The finding of PVS-like hyperintensities on T2w MRI support the SHRSP rat's clinical relevance for studying the underlying pathophysiology of SVD.

Isolation of Primary Human and Rodent Brain Microvascular Endothelial Cells: Culturing, Characterization, and High-Efficiency Transfection.

Studies of blood-brain barrier (BBB) require developing of a novel and convenient in vitro endothelial cell model. We isolated primary human and rodent brain microvascular endothelial cells and developed methods for culturing, characterization, and high-efficiency transfection of endothelial cells. Here, we describe the improved methods to obtain in vitro human and rodent BBB models to study expression of endogenous and exogenous genes of interest.