Role of PFKFB3-driven glycolysis in vessel sprouting.

Vessel sprouting by migrating tip and proliferating stalk endothelial cells (ECs) is controlled by genetic signals (such as Notch), but it is unknown whether metabolism also regulates this process. Here, we show that ECs relied on glycolysis rather than on oxidative phosphorylation for ATP production and that loss of the glycolytic activator PFKFB3 in ECs impaired vessel formation. Mechanistically, PFKFB3 not only regulated EC proliferation but also controlled the formation of filopodia/lamellipodia and directional migration, in part by compartmentalizing with F-actin in motile protrusions. Mosaic in vitro and in vivo sprouting assays further revealed that PFKFB3 overexpression overruled the pro-stalk activity of Notch, whereas PFKFB3 deficiency impaired tip cell formation upon Notch blockade, implying that glycolysis regulates vessel branching.

Relief of hypoxia by angiogenesis promotes neural stem cell differentiation by targeting glycolysis.

Blood vessels are part of the stem cell niche in the developing cerebral cortex, but their in vivo role in controlling the expansion and differentiation of neural stem cells (NSCs) in development has not been studied. Here, we report that relief of hypoxia in the developing cerebral cortex by ingrowth of blood vessels temporo-spatially coincided with NSC differentiation. Selective perturbation of brain angiogenesis in vessel-specific Gpr124 null embryos, which prevented the relief from hypoxia, increased NSC expansion at the expense of differentiation. Conversely, exposure to increased oxygen levels rescued NSC differentiation in Gpr124 null embryos and increased it further in WT embryos, suggesting that niche blood vessels regulate NSC differentiation at least in part by providing oxygen. Consistent herewith, hypoxia-inducible factor (HIF)-1α levels controlled the switch of NSC expansion to differentiation. Finally, we provide evidence that high glycolytic activity of NSCs is required to prevent their precocious differentiation in vivo Thus, blood vessel function is required for efficient NSC differentiation in the developing cerebral cortex by providing oxygen and possibly regulating NSC metabolism.

Inhibition of MicroRNA-146a and Overexpression of Its Target Dihydrolipoyl Succinyltransferase Protect Against Pressure Overload-Induced Cardiac Hypertrophy and Dysfunction.

BACKGROUND: Cardiovascular diseases remain the predominant cause of death worldwide, with the prevalence of heart failure continuing to increase. Despite increased knowledge of the metabolic alterations that occur in heart failure, novel therapies to treat the observed metabolic disturbances are still lacking. METHODS: Mice were subjected to pressure overload by means of angiotensin-II infusion or transversal aortic constriction. MicroRNA-146a was either genetically or pharmacologically knocked out or genetically overexpressed in cardiomyocytes. Furthermore, overexpression of dihydrolipoyl succinyltransferase (DLST) in the murine heart was performed by means of an adeno-associated virus. RESULTS: MicroRNA-146a was upregulated in whole heart tissue in multiple murine pressure overload models. Also, microRNA-146a levels were moderately increased in left ventricular biopsies of patients with aortic stenosis. Overexpression of microRNA-146a in cardiomyocytes provoked cardiac hypertrophy and left ventricular dysfunction in vivo, whereas genetic knockdown or pharmacological blockade of microRNA-146a blunted the hypertrophic response and attenuated cardiac dysfunction in vivo. Mechanistically, microRNA-146a reduced its target DLST-the E2 subcomponent of the α-ketoglutarate dehydrogenase complex, a rate-controlling tricarboxylic acid cycle enzyme. DLST protein levels significantly decreased on pressure overload in wild-type mice, paralleling a decreased oxidative metabolism, whereas DLST protein levels and hence oxidative metabolism were partially maintained in microRNA-146a knockout mice. Moreover, overexpression of DLST in wild-type mice protected against cardiac hypertrophy and dysfunction in vivo. CONCLUSIONS: Altogether we show that the microRNA-146a and its target DLST are important metabolic players in left ventricular dysfunction.

Evidence of amyloid-ß cerebral amyloid angiopathy transmission through neurosurgery.

Amyloid-ß (Aß) is a peptide deposited in the brain parenchyma in Alzheimer's disease and in cerebral blood vessels, causing cerebral amyloid angiopathy (CAA). Aß pathology is transmissible experimentally in animals and through medical procedures in humans, such as contaminated growth hormone or dura mater transplantation in the context of iatrogenic prion disease. Here, we present four patients who underwent neurosurgical procedures during childhood or teenage years and presented with intracerebral haemorrhage approximately three decades later, caused by severe CAA. None of these patients carried pathogenic mutations associated with early Aß pathology development. In addition, we identified in the literature four patients with a history of neurosurgical intervention and subsequent development of CAA. These findings raise the possibility that Aß pathology may be transmissible, as prion disease is, through neurosurgical procedures.

Cleaved TMEM106B forms amyloid aggregates in central and peripheral nervous systems.

Filaments made of residues 120-254 of transmembrane protein 106B (TMEM106B) form in an age-dependent manner and can be extracted from the brains of neurologically normal individuals and those of subjects with a variety of neurodegenerative diseases. TMEM106B filament formation requires cleavage at residue 120 of the 274 amino acid protein; at present, it is not known if residues 255-274 form the fuzzy coat of TMEM106B filaments. Here we show that a second cleavage appears likely, based on staining with an antibody raised against residues 263-274 of TMEM106B. We also show that besides the brain TMEM106B inclusions form in dorsal root ganglia and spinal cord, where they were mostly found in non-neuronal cells. We confirm that in the brain, inclusions were most abundant in astrocytes. No inclusions were detected in heart, liver, spleen or hilar lymph nodes. Based on their staining with luminescent conjugated oligothiophenes, we confirm that TMEM106B inclusions are amyloids. By in situ immunoelectron microscopy, TMEM106B assemblies were often found in structures resembling endosomes and lysosomes.

The annotation of GBA1 has been concealed by its protein-coding pseudogene GBAP1.

Mutations in GBA1 cause Gaucher disease and are the most important genetic risk factor for Parkinson's disease. However, analysis of transcription at this locus is complicated by its highly homologous pseudogene, GBAP1. We show that >50% of short RNA-sequencing reads mapping to GBA1 also map to GBAP1. Thus, we used long-read RNA sequencing in the human brain, which allowed us to accurately quantify expression from both GBA1 and GBAP1. We discovered significant differences in expression compared to short-read data and identify currently unannotated transcripts of both GBA1 and GBAP1. These included protein-coding transcripts from both genes that were translated in human brain, but without the known lysosomal function-yet accounting for almost a third of transcription. Analyzing brain-specific cell types using long-read and single-nucleus RNA sequencing revealed region-specific variations in transcript expression. Overall, these findings suggest nonlysosomal roles for GBA1 and GBAP1 with implications for our understanding of the role of GBA1 in health and disease.

Benefits for children with suspected cancer from routine whole-genome sequencing.

Clinical whole-genome sequencing (WGS) has been shown to deliver potential benefits to children with cancer and to alter treatment in high-risk patient groups. It remains unknown whether offering WGS to every child with suspected cancer can change patient management. We collected WGS variant calls and clinical and diagnostic information from 281 children (282 tumors) across two English units (n = 152 from a hematology center, n = 130 from a solid tumor center) where WGS had become a routine test. Our key finding was that variants uniquely attributable to WGS changed the management in ~7% (20 out of 282) of cases while providing additional disease-relevant findings, beyond standard-of-care molecular tests, in 108 instances for 83 (29%) cases. Furthermore, WGS faithfully reproduced every standard-of-care molecular test (n = 738) and revealed several previously unknown genomic features of childhood tumors. We show that WGS can be delivered as part of routine clinical care to children with suspected cancer and can change clinical management by delivering unexpected genomic insights. Our experience portrays WGS as a clinically impactful assay for routine practice, providing opportunities for assay consolidation and for delivery of molecularly informed patient care.

Accurate digital quantification of tau pathology in progressive supranuclear palsy.

The development of novel treatments for Progressive Supranuclear Palsy (PSP) is hindered by a knowledge gap of the impact of neurodegenerative neuropathology on brain structure and function. The current standard practice for measuring postmortem tau histology is semi-quantitative assessment, which is prone to inter-rater variability, time-consuming and difficult to scale. We developed and optimized a tau aggregate type-specific quantification pipeline for cortical and subcortical regions, in human brain donors with PSP. We quantified 4 tau objects ('neurofibrillary tangles', 'coiled bodies', 'tufted astrocytes', and 'tau fragments') using a probabilistic random forest machine learning classifier. The tau pipeline achieved high classification performance (F1-score > 0.90), comparable to neuropathologist inter-rater reliability in the held-out test set. Using 240 AT8 slides from 32 postmortem brains, the tau burden was correlated against the PSP pathology staging scheme using Spearman's rank correlation. We assessed whether clinical severity (PSP rating scale, PSPRS) score reflects neuropathological severity inferred from PSP stage and tau burden using Bayesian linear mixed regression. Tufted astrocyte density in cortical regions and coiled body density in subcortical regions showed the highest correlation to PSP stage (r = 0.62 and r = 0.38, respectively). Using traditional manual staging, only PSP patients in stage 6, not earlier stages, had significantly higher clinical severity than stage 2. Cortical tau density and neurofibrillary tangle density in subcortical regions correlated with clinical severity. Overall, our data indicate the potential for highly accurate digital tau aggregate type-specific quantification for neurodegenerative tauopathies; and the importance of studying tau aggregate type-specific burden in different brain regions as opposed to overall tau, to gain insights into the pathogenesis and progression of tauopathies.

LINE-1 retrotransposons drive human neuronal transcriptome complexity and functional diversification.

The genetic mechanisms underlying the expansion in size and complexity of the human brain remain poorly understood. Long interspersed nuclear element-1 (L1) retrotransposons are a source of divergent genetic information in hominoid genomes, but their importance in physiological functions and their contribution to human brain evolution are largely unknown. Using multiomics profiling, we here demonstrate that L1 promoters are dynamically active in the developing and the adult human brain. L1s generate hundreds of developmentally regulated and cell type-specific transcripts, many that are co-opted as chimeric transcripts or regulatory RNAs. One L1-derived long noncoding RNA, LINC01876, is a human-specific transcript expressed exclusively during brain development. CRISPR interference silencing of LINC01876 results in reduced size of cerebral organoids and premature differentiation of neural progenitors, implicating L1s in human-specific developmental processes. In summary, our results demonstrate that L1-derived transcripts provide a previously undescribed layer of primate- and human-specific transcriptome complexity that contributes to the functional diversification of the human brain.

Oxygen sensing: a common crossroad in cancer and neurodegeneration.

Prolyl hydroxylase domain (PHD) proteins are cellular oxygen sensors that orchestrate an adaptive response to hypoxia and oxidative stress, executed by hypoxia-inducible factors (HIFs). By increasing oxygen supply, reducing oxygen consumption, and reprogramming metabolism, the PHD/HIF pathway confers tolerance towards hypoxic and oxidative stress. This review discusses the involvement of the PHD/HIF response in two, at first sight, entirely distinct pathologies with opposite outcome, i.e. cancer leading to cellular growth and neurodegeneration resulting in cell death. However, these disorders share common mechanisms of sensing oxygen and oxidative stress. We will focus on how PHD/HIF signaling is pathogenetically implicated in metabolic and vessel alterations in these diseases and how manipulation of this pathway might offer novel treatment opportunities.

Malignant intracerebral nerve sheath tumor in a patient with Noonan syndrome: illustrative case.

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNSTs) within the neuroaxis are rare, usually arising from peripheral and cranial nerves. Even more scarce are cranial subclassifications of MPNSTs termed "malignant intracerebral nerve sheath tumors" (MINSTs). These tumors are aggressive, with a strong tendency for metastasis. With this presentation, alongside resistance to adjunctive therapy, complete excision is the mainstay of treatment, although it is often insufficient, resulting in a high rate of mortality. OBSERVATIONS: The authors report the case of an adult patient with a history of Noonan syndrome (NS) presenting with slowly progressive right-sided hemiparesis and right-sided focal motor seizures. Despite initial imaging and histology suggesting a left frontal lobe high-grade intrinsic tumor typical of a glioblastoma, subsequent molecular analysis confirmed a diagnosis of MINST. The patient's neurological condition improved after gross-total resection and adjuvant chemo-radiation; he remains on follow-up. LESSONS: MINSTs are rare neoplasms with a poor prognosis; management options are limited, with surgery being the cornerstone of treatment. Reports on rare tumors such as this will increase awareness of this particular pathology and disclose clinical experience. In this case, the authors were unable to establish a definite cause-and-effect relation between NS and MINST. Nevertheless, it remains the first reported case in the literature.

Soluble and insoluble dipeptide repeat protein measurements in C9orf72-frontotemporal dementia brains show regional differential solubility and correlation of poly-GR with clinical severity.

A C9orf72 repeat expansion is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis. One of the suggested pathomechanisms is toxicity from dipeptide repeat proteins (DPRs), which are generated via unconventional translation of sense and antisense repeat transcripts with poly-GA, poly-GP and poly-GR being the most abundant dipeptide proteins. Animal and cellular studies highlight a neurotoxic role of poly-GR and poly-PR and to a lesser degree of poly-GA. Human post-mortem studies in contrast have been much less clear on a potential role of DPR toxicity but have largely focused on immunohistochemical methods to detect aggregated DPR inclusions. This study uses protein fractionation and sensitive immunoassays to quantify not only insoluble but also soluble poly-GA, poly-GP and poly-GR concentrations in brain homogenates of FTD patients with C9orf72 mutation across four brain regions. We show that soluble DPRs are less abundant in clinically affected areas (i.e. frontal and temporal cortices). In contrast, the cerebellum not only shows the largest DPR load but also the highest relative DPR solubility. Finally, poly-GR levels and poly-GP solubility correlate with clinical severity. These findings provide the first cross-comparison of soluble and insoluble forms of all sense DPRs and shed light on the distribution and role of soluble DPRs in the etiopathogenesis of human C9orf72-FTD.

Cerebrospinal fluid cannot be used to distinguish inflammatory myelitis from congestive myelopathy due to spinal dural arteriovenous fistula: case series.

Patients with congestive myelopathy due to spinal dural arteriovenous fistula (SDAVF) typically present with progressive sensory and motor disturbance in association with sphincter dysfunction. Spinal MRI usually shows longitudinally extensive T2 signal change. Here, we report four patients with progressive myelopathy due to SDAVF who also presented with findings on cerebrospinal fluid (CSF) examination suggestive of an inflammatory aetiology. Such CSF findings in SDAVF are important to recognise, to avoid the erroneous diagnosis of an inflammatory myelitis and inappropriate treatment with immunosuppression. SDAVF can be difficult to detect and may require repeated investigation, with formal angiography as the gold standard.

Differentiation but not ALS mutations in FUS rewires motor neuron metabolism.

Energy metabolism has been repeatedly linked to amyotrophic lateral sclerosis (ALS). Yet, motor neuron (MN) metabolism remains poorly studied and it is unknown if ALS MNs differ metabolically from healthy MNs. To address this question, we first performed a metabolic characterization of induced pluripotent stem cells (iPSCs) versus iPSC-derived MNs and subsequently compared MNs from ALS patients carrying FUS mutations to their CRISPR/Cas9-corrected counterparts. We discovered that human iPSCs undergo a lactate oxidation-fuelled prooxidative metabolic switch when they differentiate into functional MNs. Simultaneously, they rewire metabolic routes to import pyruvate into the TCA cycle in an energy substrate specific way. By comparing patient-derived MNs and their isogenic controls, we show that ALS-causing mutations in FUS did not affect glycolytic or mitochondrial energy metabolism of human MNs in vitro. These data show that metabolic dysfunction is not the underlying cause of the ALS-related phenotypes previously observed in these MNs.

The spectrum of epilepsy caused by POLG mutations.

Mutations in POLG are increasingly recognized as a cause of refractory occipital lobe epilepsy (OLE) and status epilepticus (SE). Our aim was to describe the epilepsy syndrome in seven patients with POLG mutations. We retrospectively reviewed the medical records of seven patients with POLG mutations and epilepsy. Mutation analysis was performed by direct sequencing of the coding exons of the POLG gene. Disease onset was at a median age of 18 years (range 12-26). Epilepsy was the presenting problem in six patients. All had focal seizures, with motor (n = 6) and visual (n = 6) phenomena. Six patients had secondarily generalized seizures and two patients had myoclonic seizures. Six patients had one or more episodes of refractory SE, including focal (n = 5), subtle (n = 4), myoclonic (n = 2) and convulsive (n = 3) SE. During or after SE, brain MRI showed lesions affecting the occipital lobe in all patients, probably due to continuous epileptic activity. Five of the six patients with SE died during treatment of SE, one due to valproate-induced hepatotoxicity. Associated clinical symptoms were ataxia (n = 6), polyneuropathy (n = 6), progressive external ophthalmoplegia (PEO) (n = 3) and migraine (n = 3). Epilepsy may be the first and dominant neurological problem caused by POLG mutations. The epilepsy may be severe and the condition of the patient may end in fatal SE. Refractory OLE and SE in a patient with polyneuropathy, ataxia, PEO or migraine warrant screening for POLG mutations. In this clinical setting, valproate should not be given in view of the risk of fatal hepatotoxicity.

Deletion or Inhibition of the Oxygen Sensor PHD1 Protects against Ischemic Stroke via Reprogramming of Neuronal Metabolism.

The oxygen-sensing prolyl hydroxylase domain proteins (PHDs) regulate cellular metabolism, but their role in neuronal metabolism during stroke is unknown. Here we report that PHD1 deficiency provides neuroprotection in a murine model of permanent brain ischemia. This was not due to an increased collateral vessel network. Instead, PHD1(-/-) neurons were protected against oxygen-nutrient deprivation by reprogramming glucose metabolism. Indeed, PHD1(-/-) neurons enhanced glucose flux through the oxidative pentose phosphate pathway by diverting glucose away from glycolysis. As a result, PHD1(-/-) neurons increased their redox buffering capacity to scavenge oxygen radicals in ischemia. Intracerebroventricular injection of PHD1-antisense oligonucleotides reduced the cerebral infarct size and neurological deficits following stroke. These data identify PHD1 as a regulator of neuronal metabolism and a potential therapeutic target in ischemic stroke.

Incomplete and transitory decrease of glycolysis: a new paradigm for anti-angiogenic therapy?

During vessel sprouting, a migratory endothelial tip cell guides the sprout, while proliferating stalk cells elongate the branch. Tip and stalk cell phenotypes are not genetically predetermined fates, but are dynamically interchangeable to ensure that the fittest endothelial cell (EC) leads the vessel sprout. ECs increase glycolysis when forming new blood vessels. Genetic deficiency of the glycolytic activator PFKFB3 in ECs reduces vascular sprouting by impairing migration of tip cells and proliferation of stalk cells. PFKFB3-driven glycolysis promotes the tip cell phenotype during vessel sprouting, since PFKFB3 overexpression overrules the pro-stalk activity of Notch signaling. Furthermore, PFKFB3-deficient ECs cannot compete with wild-type neighbors to form new blood vessels in chimeric mosaic mice. In addition, pharmacological PFKFB3 blockade reduces pathological angiogenesis with modest systemic effects, likely because it decreases glycolysis only partially and transiently.

Partial and transient reduction of glycolysis by PFKFB3 blockade reduces pathological angiogenesis.

Strategies targeting pathological angiogenesis have focused primarily on blocking vascular endothelial growth factor (VEGF), but resistance and insufficient efficacy limit their success, mandating alternative antiangiogenic strategies. We recently provided genetic evidence that the glycolytic activator phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3) promotes vessel formation but did not explore the antiangiogenic therapeutic potential of PFKFB3 blockade. Here, we show that blockade of PFKFB3 by the small molecule 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) reduced vessel sprouting in endothelial cell (EC) spheroids, zebrafish embryos, and the postnatal mouse retina by inhibiting EC proliferation and migration. 3PO also suppressed vascular hyperbranching induced by inhibition of Notch or VEGF receptor 1 (VEGFR1) and amplified the antiangiogenic effect of VEGF blockade. Although 3PO reduced glycolysis only partially and transiently in vivo, this sufficed to decrease pathological neovascularization in ocular and inflammatory models. These insights may offer therapeutic antiangiogenic opportunities.

Tumor vessel normalization by chloroquine independent of autophagy.

Chloroquine (CQ) has been evaluated as an autophagy blocker for cancer treatment, but it is unknown if it acts solely by inhibiting cancer cell autophagy. We report that CQ reduced tumor growth but improved the tumor milieu. By normalizing tumor vessel structure and function and increasing perfusion, CQ reduced hypoxia, cancer cell invasion, and metastasis, while improving chemotherapy delivery and response. Inhibiting autophagy in cancer cells or endothelial cells (ECs) failed to induce such effects. CQ's vessel normalization activity relied mainly on alterations of endosomal Notch1 trafficking and signaling in ECs and was abrogated by Notch1 deletion in ECs in vivo. Thus, autophagy-independent vessel normalization by CQ restrains tumor invasion and metastasis while improving chemotherapy, supporting the use of CQ for anticancer treatment.

The neurovascular link in health and disease: molecular mechanisms and therapeutic implications.

At first sight, the nervous and vascular systems seem to share little in common. However, neural and vascular cells not only are anatomically closely tied to each other, but they also utilize and respond to similar classes of signals to establish correct connectivity and wiring of their networks. Recent studies further provide evidence that this neurovascular crosstalk is more important for understanding the molecular basis of neurological disease than originally anticipated. Moreover, neurovascular strategies offer novel therapeutic opportunities for neurodegenerative disorders.