Loss of the neuron-specific F-box protein FBXO41 models an ataxia-like phenotype in mice with neuronal migration defects and degeneration in the cerebellum.
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FBXO41 is a novel CNS/neuron-specific F-box protein that localizes to the centrosome and cytoplasm of neurons, and cytoplasmic FBXO41 promotes neuronal migration.
"we identify FBXO41 as a novel CNS-specific F-box protein that localizes to the centrosome and the cytoplasm of neurons and demonstrate that cytoplasmic FBXO41 promotes neuronal migration"
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FBXO41 protein is restricted to CNS tissues (cortex, hippocampus, cerebellum, spinal cord), absent from peripheral nerve and non-neural tissues, detectable from embryonic day 10, and expressed in cultured granule neurons but not astrocytes or oligodendrocytes.
"We found FBXO41 to be expressed in CNS tissues including cortex, hippocampus, cerebellum, and spinal cord, but absent from the peripheral nervous system tissue such as sciatic nerve and from non-neural tissues"
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Endogenous FBXO41 is cytoplasmic and excluded from the nucleus, with a centrosomal pool confirmed by GFP-FBXO41 colocalization with pericentrin 2 and cosegregation of endogenous FBXO41 with gamma-tubulin in centrosome purifications.
"we discovered a conspicuous dot-like accumulation of FBXO41 near the nucleus, which we later confirmed to be the centrosome because GFP-FBXO41 colocalized with the centrosomal marker pericentrin 2"
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A centrosomal targeting region (CTR, aa 179-354) directs FBXO41 to the centrosome; FBXO41 lacking the CTR fails to localize to the centrosome and disperses in the cytoplasm, establishing dual centrosomal/cytoplasmic localization.
"we refer to it as centrosomal targeting region (CTR, aa 179–354) hereafter"
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In vivo electroporation knockdown of FBXO41 in P4 rat cerebellum stalls granule neurons in the EGL/molecular layer with fewer reaching the inner IGL, indicating slower migration; FBXO41 promotes neuronal migration.
"we found that significantly more FBXO41 knock-down neurons were stalled in the EGL/ML"
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Rescue with the cytoplasm-only FBXO41 (lacking the CTR), but not centrosome-targeted forms, restores migration, and FBXO41 gain-of-function accelerates migration into the inner IGL, showing the cytoplasmic pool is the crucial one for migration.
"Collectively, these data indicate that the FBXO41 RNAi-induced phenotype is specific to FBXO41 and suggest that cytoplasmic FBXO41 is crucial to stimulate the migration of cerebellar granule neurons"
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FBXO41-knockout mice have high perinatal lethality and at P16 display a severely ataxic gait with hind-limb dragging, impaired balance, tremors, and hindlimb clasping.
"mice displayed an abnormal gait characterized by dragging of the hind limbs, impaired balance, and tremors"
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The FBXO41-knockout cerebellum shows delayed granule-neuron migration (residual EGL and excess molecular-layer cells across P12-P30); migrating cells eventually reach the IGL, so FBXO41 sets migration speed rather than being strictly essential.
"although the migrating cells eventually catch up and reach the IGL, FBXO41 is required to maintain a normal migration speed in the cerebellum"
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Neurodegeneration in the knockout is age-dependent, absent at P16 but by P30 showing a significant increase in apoptotic (TUNEL+) cells in hippocampus and cerebellum, alongside a distorted, smaller cerebellum.
"found little or no increase of apoptotic cells in the cortex but a significant increase in the hippocampus and the cerebellum"
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FBXO41 has a cell-autonomous prosurvival role in neurons; functional RNAi knockdown increases cleaved-caspase-3+ granule neurons, rescued by RNAi-resistant FBXO41.
"These results support a prosurvival role for FBXO41 in neurons."