Ocular pterygium-digital keloid dysplasia (OPDKD) presents in childhood with ingrowth of vascularized connective tissue on the cornea leading to severely reduced vision. Later the patients develop keloids on digits but are otherwise healthy. The overgrowth in OPDKD affects body parts that typically have lower temperature than 37°C. We present evidence that OPDKD is associated with a temperature sensitive, activating substitution, p.(Asn666Tyr), in PDGFRB. Phosphorylation levels of PDGFRB and downstream targets were higher in OPDKD fibroblasts at 37°C but were further greatly increased at the average corneal temperature of 32°C. This suggests that the substitution cause significant constitutive autoactivation mainly at lower temperature. In contrast, a different substitution in the same codon, p.(Asn666Ser), is associated with Penttinen type of premature aging syndrome. This devastating condition is characterized by widespread tissue degeneration, including pronounced chronic ulcers and osteolytic resorption in distal limbs. In Penttinen syndrome fibroblasts, equal and high levels of phosphorylated PDGFRB was present at both 32°C and 37°C. This indicates that this substitution causes severe constitutive autoactivation of PDGFRB regardless of temperature. In line with this, most downstream targets were not affected by lower temperature. However, STAT1, important for tissue wasting, did show further increased phosphorylation at 32°C. Temperature-dependent autoactivation offers an explanation to the strikingly different clinical outcomes of substitutions in the Asn666 codon of PDGFRB.

Biallelic Parkin (PRKN) mutations cause autosomal recessive Parkinson's disease (PD); however, the role of monoallelic PRKN mutations as a risk factor for PD remains unclear. We investigated the role of single heterozygous PRKN mutations in three large independent case-control cohorts totalling 10 858 PD cases and 8328 controls. Overall, after exclusion of biallelic carriers, single PRKN mutations were more common in PD than controls conferring a >1.5-fold increase in the risk of PD [P-value (P) = 0.035], with meta-analysis (19 574 PD cases and 468 488 controls) confirming increased risk [Odds ratio (OR) = 1.65, P = 3.69E-07]. Carriers were shown to have significantly younger ages at the onset compared with non-carriers (NeuroX: 56.4 vs. 61.4 years; exome: 38.5 vs. 43.1 years). Stratifying by mutation type, we provide preliminary evidence for a more pathogenic risk profile for single PRKN copy number variant (CNV) carriers compared with single nucleotide variant carriers. Studies that did not assess biallelic PRKN mutations or consist of predominantly early-onset cases may be biasing these estimates, and removal of these resulted in a loss of association (OR = 1.23, P = 0.614; n = 4). Importantly, when we looked for additional CNVs in 30% of PD cases with apparent monoallellic PRKN mutations, we found that 44% had biallelic mutations, suggesting that previous estimates may be influenced by cryptic biallelic mutation status. While this study supports the association of single PRKN mutations with PD, it highlights confounding effects; therefore, caution is needed when interpreting current risk estimates. Together, we demonstrate that comprehensive assessment of biallelic mutation status is essential when elucidating PD risk associated with monoallelic PRKN mutations.

In the last few years, NEK1 has been identified as a new gene related to amyotrophic lateral sclerosis (ALS). Loss-of-function variants have been mostly described, although several missense variants exist, which pathogenic relevance remains to be established. We attempted to determine the contribution of NEK1 gene in an Italian cohort of 531 sporadic and familial amyotrophic lateral sclerosis (ALS) patients applying massive parallel sequencing technologies. We filtered results of NEK1 gene and identified 20 NEK1 rare variants (MAF < 0.01) in 22 patients. In particular, we found two novel frameshift variants (p.Glu929Asnfs*12 and p.Val1030Ilefs*23), 18 missense variants, including the p.Arg261His in three patients, and a novel variant in the start codon, the p.Met1?, which most likely impairs translation initiation. To clarify the role of NEK1 missense variants we investigated NEK1 expression in primary fibroblast cultures. We obtained skin biopsies from four patients with NEK1 variants and we assessed NEK1 expression by western blot and immunofluorescence. We detected a decrease in NEK1 expression in fibroblasts from patients with NEK1 variants, suggesting that missense variants in NEK1 gene may have a pathogenic role. Moreover, we observed additional variants in ALS related genes in seven patients with NEK1 variants (32%), further supporting an oligogenic ALS model.

Leber's hereditary optic neuropathy (LHON) is the most common disorder due to mitochondrial DNA mutations and complex I deficiency. It is characterized by an acute vision loss, generally in young adults, with a higher penetrance in males. How complex I dysfunction induces the peculiar LHON clinical presentation remains an unanswered question. To gain an insight into this question, we carried out a non-targeted metabolomic investigation using the plasma of 18 LHON patients, during the chronic phase of the disease, comparing them to 18 healthy controls. A total of 500 metabolites were screened of which 156 were accurately detected. A supervised Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) highlighted a robust model for disease prediction with a Q2 (cum) of 55.5%, with a reliable performance during the permutation test (cross-validation analysis of variance, P-value = 5.02284e-05) and a good prediction of a test set (P = 0.05). This model highlighted 10 metabolites with variable importance in the projection (VIP) > 0.8. Univariate analyses revealed nine discriminating metabolites, six of which were the same as those found in the Orthogonal Projections to Latent Structures Discriminant Analysis model. In total, the 13 discriminating metabolites identified underlining dietary metabolites (nicotinamide, taurine, choline, 1-methylhistidine and hippurate), mitochondrial energetic substrates (acetoacetate, glutamate and fumarate) and purine metabolism (inosine). The decreased concentration of taurine and nicotinamide (vitamin B3) suggest interesting therapeutic targets, given their neuroprotective roles that have already been demonstrated for retinal ganglion cells. Our results show a reliable predictive metabolomic signature in the plasma of LHON patients and highlighted taurine and nicotinamide deficiencies.

Glioblastoma (GB) is the most frequent and malignant type of brain tumor, for which no effective therapy exists. The high proliferative and invasive nature of GB, as well as its acquired resistance to chemotherapy, makes this type of cancer extremely lethal shortly after diagnosis. Long non-protein coding RNAs (lncRNA) are a class of regulatory RNAs whose levels can be dysregulated in the context of diseases, unbalancing several physiological processes. The lncRNA associated with microvascular invasion in hepatocellular carcinoma (lncRNA-MVIH), overexpressed in several cancers, was described to co-precipitate with phosphoglycerate kinase 1 (PGK1), preventing secretion of this enzyme to the extracellular environment and promoting cell migration and invasion. We hypothesized that, by silencing the expression of lncRNA-MVIH, the secretion of PGK1 would increase, reducing GB cell migration and invasion capabilities. We observed that lncRNA-MVIH silencing in human GB cells significantly decreased glycolysis, cell growth, migration, and invasion and sensitized GB cells to cediranib. However, no increase in extracellular PGK1 was observed as a consequence of lncRNA-MVIH silencing, and therefore, we investigated the possibility of a mechanism of miRNA sponge of lncRNA-MVIH being in place. We found that the levels of miR-302a loaded onto RISC increased in GB cells after lncRNA-MVIH silencing, with the consequent downregulation of several miR-302a molecular targets. Our findings suggest a new mechanism of action of lncRNA-MVIH as a sponge of miR-302a. We suggest that lncRNA-MVIH knockdown may be a promising strategy to address GB invasiveness and chemoresistance, holding potential towards its future application in a clinical context.

GSTP proteins are metabolic enzymes involved in the removal of oxidative stress and intracellular signaling and also have inhibitory effects on JNK activity. However, the functions of Gstp proteins in the developing brain are unknown. In mice, there are three Gstp proteins, Gstp1, 2 and 3, whereas there is only one GSTP in humans. By reverse transcription-polymerase chain reaction (RT-PCR) analysis, we found that Gstp1 was expressed beginning at E15.5 in the cortex, but Gstp2 and 3 started expressing at E18.5. Gstp 1 and 2 knockdown (KD) caused decreased neurite number in cortical neurons, implicating them in neurite initiation. Using in utero electroporation (IUE) to knock down Gstp1 and 2 in layer 2/3 pyramidal neurons in vivo, we found abnormal swelling of the apical dendrite at P3 and reduced neurite number at P15. Using time-lapse live imaging, we found that the apical dendrite orientation was skewed compared with the control. We explored the molecular mechanism and found that JNK inhibition rescued reduced neurite number caused by Gstp knockdown, indicating that Gstp regulates neurite formation through JNK signaling. Thus, we found novel functions of Gstp proteins in neurite initiation during cortical development. These findings not only provide novel functions of Gstp proteins in neuritogenesis during cortical development but also help us to understand the complexity of neurite formation.

Oligodendrocytes exist in a heterogenous state and are implicated in multiple neuropsychiatric diseases including dementia. Cortical oligodendrocytes are a glial population uniquely positioned to play a key role in neurodegeneration by synchronizing circuit connectivity but molecular pathways specific to this role are lacking. We utilized oligodendrocyte-specific translating ribosome affinity purification and RNA-seq (TRAP-seq) to transcriptionally profile adult mature oligodendrocytes from different regions of the central nervous system. Weighted gene co-expression network analysis reveals distinct region-specific gene networks. Two of these mature myelinating oligodendrocyte gene networks uniquely define cortical oligodendrocytes and differentially regulate cortical myelination (M8) and synaptic signaling (M4). These two cortical oligodendrocyte gene networks are enriched for genes associated with dementia including MAPT and include multiple gene targets of the regulatory microRNA, miR-142-3p. Using a combination of TRAP-qPCR, miR-142-3p overexpression in vitro, and miR-142-null mice, we show that miR-142-3p negatively regulates cortical myelination. In rTg4510 tau-overexpressing mice, cortical myelination is compromised, and tau-mediated neurodegeneration is associated with gene co-expression networks that recapitulate both the M8 and M4 cortical oligodendrocyte gene networks identified from normal cortex. We further demonstrate overlapping gene networks in mature oligodendrocytes present in normal cortex, rTg4510 and miR-142-null mice, and existing datasets from human tauopathies to provide evidence for a critical role of miR-142-3p-regulated cortical myelination and oligodendrocyte-mediated synaptic signaling in neurodegeneration.

The BBSome is a protein complex consisting of BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9 and BBS18 that associates with intraflagellar transport complexes and specializes in ciliary trafficking. In primary cilia, ciliary entry requires the fully assembled BBSome as well as the small GTPase, ARL6 (BBS3). Retinal photoreceptors possess specialized cilia. In light of key structural and functional differences between primary and specialized cilia, we examined the principles of BBSome recruitment to photoreceptor cilia. We performed sucrose gradient fractionation using retinal lysates of Bbs2-/-, Bbs7-/-, Bbs8-/- and Bbs3-/- mice to determine the status of BBSome assembly, then determined localization of BBSome components using immunohistochemistry. Surprisingly, we found that a subcomplex of the BBSome containing at least BBS1, BBS5, BBS8 and BBS9 is recruited to cilia in the absence of BBS2 or BBS7. In contrast, a BBSome subcomplex consisting of BBS1, BBS2, BBS5, BBS7 and BBS9 is found in Bbs8-/- retinas and is denied ciliary entry in photoreceptor cells. In addition, the BBSome remains fully assembled in Bbs3-/- retinas and can be recruited to photoreceptor cilia in the absence of BBS3. We compared phenotypic severity of their retinal degeneration phenotypes. These findings demonstrate that unlike primary cilia, photoreceptor cilia admit a partially assembled BBSome meeting specific requirements. In addition, the recruitment of the BBSome to photoreceptor cilia does not require BBS3. These findings indicate that the ciliary entry of the BBSome is subjected to cell-specific regulation, particularly in cells with highly adapted forms of cilia such as photoreceptors.

DNA methylation (DNAm) is known to play a pivotal role in childhood health and development, but a comprehensive characterization of genome-wide DNAm trajectories across this age period is currently lacking. We have therefore performed a series of epigenome-wide association studies in 5019 blood samples collected at multiple time-points from birth to late adolescence from 2348 participants of two large independent cohorts. DNAm profiles of autosomal CpG sites (CpGs) were generated using the Illumina Infinium HumanMethylation450 BeadChip. Change over time was widespread, observed at over one-half (53%) of CpGs. In most cases, DNAm was decreasing (36% of CpGs). Inter-individual variation in linear trajectories was similarly widespread (27% of CpGs). Evidence for non-linear change and inter-individual variation in non-linear trajectories was somewhat less common (11 and 8% of CpGs, respectively). Very little inter-individual variation in change was explained by sex differences (0.4% of CpGs) even though sex-specific DNAm was observed at 5% of CpGs. DNAm trajectories were distributed non-randomly across the genome. For example, CpGs with decreasing DNAm were enriched in gene bodies and enhancers and were annotated to genes enriched in immune-developmental functions. In contrast, CpGs with increasing DNAm were enriched in promoter regions and annotated to genes enriched in neurodevelopmental functions. These findings depict a methylome undergoing widespread and often non-linear change throughout childhood. They support a developmental role for DNA methylation that extends beyond birth into late adolescence and has implications for understanding life-long health and disease. DNAm trajectories can be visualized at http://epidelta.mrcieu.ac.uk.

FEZ1-mediated axonal transport plays important roles in central nervous system development but its involvement in the peripheral nervous system is not well-characterized. FEZ1 is deleted in Jacobsen syndrome (JS), an 11q terminal deletion developmental disorder. JS patients display impaired psychomotor skills, including gross and fine motor delay, suggesting that FEZ1 deletion may be responsible for these phenotypes, given its association with the development of motor-related circuits. Supporting this hypothesis, our data show that FEZ1 is selectively expressed in the rat brain and spinal cord. Its levels progressively increase over the developmental course of human motor neurons (MN) derived from embryonic stem cells. Deletion of FEZ1 strongly impaired axon and dendrite development, and significantly delayed the transport of synaptic proteins into developing neurites. Concurring with these observations, Drosophila unc-76 mutants showed severe locomotion impairments, accompanied by a strong reduction of synaptic boutons at neuromuscular junctions. These abnormalities were ameliorated by pharmacological activation of UNC-51/ATG1, a FEZ1-activating kinase, with rapamycin and metformin. Collectively, the results highlight a role for FEZ1 in MN development and implicate its deletion as an underlying cause of motor impairments in JS patients.