Hemispheric Asymmetry in New Neurons in Adulthood Is Associated with Vocal Learning and Auditory Memory

Many brain regions exhibit lateral differences in structure and function, and also incorporate new neurons in adulthood, thought to function in learning and in the formation of new memories. However, the contribution of new neurons to hemispheric differences in processing is unknown. The present study combines cellular, behavioral, and physiological methods to address whether 1) new neuron incorporation differs between the brain hemispheres, and 2) the degree to which hemispheric lateralization of new neurons correlates with behavioral and physiological measures of learning and memory. The songbird provides a model system for assessing the contribution of new neurons to hemispheric specialization because songbird brain areas for vocal processing are functionally lateralized and receive a continuous influx of new neurons in adulthood. In adult male zebra finches, we quantified new neurons in the caudomedial nidopallium (NCM), a forebrain area involved in discrimination and memory for the complex vocalizations of individual conspecifics. We assessed song learning and recorded neural responses to song in NCM. We found significantly more new neurons labeled in left than in right NCM; moreover, the degree of asymmetry in new neuron numbers was correlated with the quality of song learning and strength of neuronal memory for recently heard songs. In birds with experimentally impaired song quality, the hemispheric difference in new neurons was diminished. These results suggest that new neurons may contribute to an allocation of function between the hemispheres that underlies the learning and processing of complex signals

From methylation to myelination: epigenomic and transcriptomic profiling of chronic inactive demyelinated multiple sclerosis lesions.

In the progressive phase of multiple sclerosis (MS), the hampered differentiation capacity of oligodendrocyte precursor cells (OPCs) eventually results in remyelination failure. We have previously shown that DNA methylation of Id2/Id4 is highly involved in OPC differentiation and remyelination. In this study, we took an unbiased approach by determining genome-wide DNA methylation patterns within chronically demyelinated MS lesions and investigated how certain epigenetic signatures relate to OPC differentiation capacity. We compared genome-wide DNA methylation and transcriptional profiles between chronically demyelinated MS lesions and matched normal-appearing white matter (NAWM), making use of post-mortem brain tissue (n = 9/group). DNA methylation differences that inversely correlated with mRNA expression of their corresponding genes were validated for their cell-type specificity in laser-captured OPCs using pyrosequencing. The CRISPR-dCas9-DNMT3a/TET1 system was used to epigenetically edit human-iPSC-derived oligodendrocytes to assess the effect on cellular differentiation. Our data show hypermethylation of CpGs within genes that cluster in gene ontologies related to myelination and axon ensheathment. Cell type-specific validation indicates a region-dependent hypermethylation of MBP, encoding for myelin basic protein, in OPCs obtained from white matter lesions compared to NAWM-derived OPCs. By altering the DNA methylation state of specific CpGs within the promotor region of MBP, using epigenetic editing, we show that cellular differentiation and myelination can be bidirectionally manipulated using the CRISPR-dCas9-DNMT3a/TET1 system in vitro. Our data indicate that OPCs within chronically demyelinated MS lesions acquire an inhibitory phenotype, which translates into hypermethylation of crucial myelination-related genes. Altering the epigenetic status of MBP can restore the differentiation capacity of OPCs and possibly boost (re)myelination

Selective PDE4 subtype inhibition provides new opportunities to intervene in neuroinflammatory versus myelin damaging hallmarks of multiple sclerosis.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterized by focal inflammatory lesions and prominent demyelination. Even though the currently available therapies are effective in treating the initial stages of disease, they are unable to halt or reverse disease progression into the chronic progressive stage. Thus far, no repair-inducing treatments are available for progressive MS patients. Hence, there is an urgent need for the development of new therapeutic strategies either targeting the destructive immunological demyelination or boosting endogenous repair mechanisms. Using in vitro, ex vivo, and in vivo models, we demonstrate that selective inhibition of phosphodiesterase 4 (PDE4), a family of enzymes that hydrolyzes and inactivates cyclic adenosine monophosphate (cAMP), reduces inflammation and promotes myelin repair. More specifically, we segregated the myelination-promoting and anti-inflammatory effects into a PDE4D- and PDE4B-dependent process respectively. We show that inhibition of PDE4D boosts oligodendrocyte progenitor cells (OPC) differentiation and enhances (re)myelination of both murine OPCs and human iPSC-derived OPCs. In addition, PDE4D inhibition promotes in vivo remyelination in the cuprizone model, which is accompanied by improved spatial memory and reduced visual evoked potential latency times. We further identified that PDE4B-specific inhibition exerts anti-inflammatory effects since it lowers in vitro monocytic nitric oxide (NO) production and improves in vivo neurological scores during the early phase of experimental autoimmune encephalomyelitis (EAE). In contrast to the pan PDE4 inhibitor roflumilast, the therapeutic dose of both the PDE4B-specific inhibitor A33 and the PDE4D-specific inhibitor Gebr32a did not trigger emesis-like side effects in rodents. Finally, we report distinct PDE4D isoform expression patterns in human area postrema neurons and human oligodendroglia lineage cells. Using the CRISPR-Cas9 system, we confirmed that pde4d1/2 and pde4d6 are the key targets to induce OPC differentiation. Collectively, these data demonstrate that gene specific PDE4 inhibitors have potential as novel therapeutic agents for targeting the distinct disease processes of MS

Mitochondrial DNA heteroplasmy distinguishes disease manifestation in PINK1/PRKN-linked Parkinson’s disease

Biallelic mutations in PINK1/PRKN cause recessive Parkinson’s disease. Given the established role of PINK1/Parkin in regulating mitochondrial dynamics, we explored mitochondrial DNA (mtDNA) integrity and inflammation as disease modifiers in carriers of mutations in these genes. MtDNA integrity was investigated in a large collection of biallelic (n = 84) and monoallelic (n = 170) carriers of PINK1/PRKN mutations, idiopathic Parkinson’s disease patients (n = 67) and controls (n = 90). In addition, we studied global gene expression and serum cytokine levels in a subset. Affected and unaffected PINK1/PRKN monoallelic mutation carriers can be distinguished by heteroplasmic mtDNA variant load (AUC = 0.83, CI:0.74-0.93). Biallelic PINK1/PRKN mutation carriers harbor more heteroplasmic mtDNA variants in blood (p = 0.0006, Z = 3.63) compared to monoallelic mutation carriers. This enrichment was confirmed in iPSC-derived (controls, n = 3; biallelic PRKN mutation carriers, n = 4) and postmortem (control, n = 1; biallelic PRKN mutation carrier, n = 1) midbrain neurons. Lastly, the heteroplasmic mtDNA variant load correlated with IL6 levels in PINK1/PRKN mutation carriers (r = 0.57, p = 0.0074). PINK1/PRKN mutations predispose individuals to mtDNA variant accumulation in a dose- and disease-dependent manner

Nanopore Single-Molecule Sequencing for Mitochondrial DNA Methylation Analysis: Investigating Parkin-Associated Parkinsonism as a Proof of Concept

Objective: To establish a workflow for mitochondrial DNA (mtDNA) CpG methylation using Nanopore whole-genome sequencing and perform first pilot experiments on affected Parkin biallelic mutation carriers (Parkin-PD) and healthy controls. Background: Mitochondria, including mtDNA, are established key players in Parkinson's disease (PD) pathogenesis. Mutations in Parkin, essential for degradation of damaged mitochondria, cause early-onset PD. However, mtDNA methylation and its implication in PD is understudied. Herein, we establish a workflow using Nanopore sequencing to directly detect mtDNA CpG methylation and compare mtDNA methylation between Parkin-related PD and healthy individuals. Methods: To obtain mtDNA, whole-genome Nanopore sequencing was performed on blood-derived from five Parkin-PD and three control subjects. In addition, induced pluripotent stem cell (iPSC)-derived midbrain neurons from four of these patients with PD and the three control subjects were investigated. The workflow was validated, using methylated and unmethylated 897 bp synthetic DNA samples at different dilution ratios (0, 50, 100% methylation) and mtDNA without methylation. MtDNA CpG methylation frequency (MF) was detected using Nanopolish and Megalodon. Results: Across all blood-derived samples, we obtained a mean coverage of 250.3X (SD ± 80.5X) and across all neuron-derived samples 830X (SD ± 465X) of the mitochondrial genome. We detected overall low-level CpG methylation from the blood-derived DNA (mean MF ± SD = 0.029 ± 0.041) and neuron-derived DNA (mean MF ± SD = 0.019 ± 0.035). Validation of the workflow, using synthetic DNA samples showed that highly methylated DNA molecules were prone to lower Guppy Phred quality scores and thereby more likely to fail Guppy base-calling. CpG methylation in blood- and neuron-derived DNA was significantly lower in Parkin-PD compared to controls (Mann-Whitney U-test p < 0.05). Conclusion: Nanopore sequencing is a useful method to investigate mtDNA methylation architecture, including Guppy-failed reads is of importance when investigating highly methylated sites. We present a mtDNA methylation workflow and suggest methylation variability across different tissues and between Parkin-PD patients and controls as an initial model to investigate

New neurons in NCM.

<p>(<b>A</b>) Medial (top, ∼170 µm from the midline) and lateral (bottom, ∼500 µm from the midline) sections of NCM (stippled area), showing the region where new neurons were quantified. Abbreviations: Cb, cerebellum; CMM, caudomedial mesopallium; HP, hippocampus; L2, Field L2; NCM, caudomedial nidopallium. Scale bar = 1 mm. (<b>B</b>) Photographs of a 1-month-old new neuron in NCM (arrow) in the same field of view showing a BrdU+ nucleus (top), Hu+ neuronal cytoplasm (middle) and co-localization of both markers (bottom). Scale bar = 10 µm.</p

Hemispheric asymmetry in new neurons.

<p>(<b>A</b>) New neuron densities are significantly correlated between the hemispheres across birds. Points above the dashed identity line are birds with higher numbers of new neurons in left than right NCM. Best-fit regression (solid orange line) intercepts the Y-axis at 283.6 (orange arrow), indicating higher neuron density on the left (n = 31; r² = 0.644; P<0.001). Red symbols are computer-tutored birds. (<b>B</b>) 1-month-old new neuron densities in NCM differ between the hemispheres. Mean new neuron density/mm³ was higher in the left NCM than the right NCM (Left = 1340.85±179.04 SEM, Right = 1025.31±139.92 SEM; paired t-test; n = 31, t = 2.999, P = 0.005, two-tailed).</p