Confined migration induces non-lethal DNA damage in developing neurons

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Owing to their limited regenerative ability, maintaining genome stability in neurons is crucial for preserving brain function throughout the lifespan 7 , 8 . However, neurons are continuously exposed to various sources of DNA damage, including intrinsic factors such as oxidative stress, transcription and neural activity, as well as extrinsic factors such as radiation and environmental toxins 9 , 10 . Neurons are therefore equipped with robust mechanisms to prevent and correct DNA lesions, as excessive or unresolved damage can contribute to brain ageing and neurodegeneration 8 , 11 , 12 .

During brain development, newborn neurons migrate from their birthplace in the germinal layers to their final destinations in the emerging cortices and nuclei, where they are integrated into functional neural circuits. Migrating neurons squeeze the nucleus, their largest cargo, through narrow spaces crowded with many other cells and extracellular components 13 , 14 . Neurons express very low levels of lamin A, which confers high nuclear deformability and migratory ability 1 , 15 , but may also render them vulnerable to mechanical stress and resulting DNA damage 2 , 16 . In cancer cells and immune cells, severe nuclear deformation during confined migration can cause transient nuclear envelope (NE) rupture and subsequent DNA damage, which has been implicated in cellular senescence and malignant transformation 3 , 4 , 5 , 6 . By contrast, the impact of nuclear deformation during neuronal migration has not been described. In this study, we investigated whether confined migration influences genome integrity in postmitotic neurons during normal brain development.

Confined migration induces DSBs in neurons

Cerebellar granule neurons (CGNs) are generated by granule neuron precursors (CGPs) in the outer external granule layer (EGL) in the cerebellar cortex (Fig. 1a ). Postmitotic CGNs move to the inner EGL and then migrate radially through the molecular layer (ML) to the internal granule layer (IGL) during the first three postnatal weeks in mice. The nuclei of migrating neurons exhibit highly dynamic motion, including frequent rotation and deformation 13 , 17 . We first studied DNA damage in the developing cerebellar cortex at postnatal day 6 (P6) using immunofluorescence analysis of γH2AX, a marker for DNA DSBs, along with the cell proliferation marker Ki-67 and the differentiated neuron marker p27 Kip1 (Fig. 1a and Extended Data Fig. 1a ). Many Ki-67-positive CGP nuclei in the outer EGL were co-stained with γH2AX. Some cells with strong γH2AX signals in the ML and IGL were identified as glial cells on the basis of BLBP expression (Extended Data Fig. 1b ). Besides these DSBs, which were presumably generated by replication stress during cell cycle progression, numerous γH2AX foci were observed in postmitotic CGNs in the ML and IGL that were co-stained with p27 Kip1 (Fig. 1a and Extended Data Fig. 1a ).

Fig. 1: Postmitotic neurons undergo DNA damage during migration through confined interstitial spaces. The alternative text for this image may have been generated using AI.

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a , Schematic of CGN differentiation during postnatal cerebellar development. CGPs (white) populate the outer EGL. CGNs (black) in the inner EGL extend axons and migrate parallel to the EGL surface and then radially through the ML to reach the IGL (top). Bottom, sagittal section of a P6 mouse cerebellar cortex stained for γH2AX (green), Ki-67 (blue) and p27 Kip1 (magenta). Postmitotic CGNs in the boxed regions in the ML and IGL bearing γH2AX foci are magnified on the right. Similar results were obtained from three mice from independent litters. b , The cerebellar cortex from P4 to P30, stained for γH2AX and p27 Kip1 . The boxed regions in the IGL are magnified at the bottom. c , The percentage of γH2AX-positive cells in the cerebellar cortical layers. Data are mean ± s.d. Two-way repeated-measures analysis of variance (ANOVA) with Dunnett’s multiple-comparison test; P values are shown. The outer EGL was excluded from the comparison. d , Snapshot image of CGNs electroporated with mScarlet (magenta) and 53BP1–mNG (green) in a cerebellar slice from a P7 mouse (left). Right, image sequence of 53BP1–mNG signals in a CGN migrating from the EGL to the ML. See also Extended Data Fig. 1e . e , f , Image sequence of a bright-field view of a CGN transfected with mScarlet and 53BP1–mNG (left) and 53BP1–mNG signals in the xy (middle) and xz (right) planes during migration ( e ), and the focus life-time in CGNs migrating in 3-µm microchannels ( f ). The box plots show the median (centre line), 25–75 percentiles (box limits), the whiskers represent the full data range, and the circle indicates the mean of 66 cells analysed. g , The percentage of CGNs that formed new 53BP1 foci (grey) or underwent NE rupture (red) during and/or after passage through channels of the indicated size. Data are mean ± s.d. Statistical analysis was performed using Welch’s ANOVA wit…