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The six subunits (Elp1 to Elp6) Elongator complex promotes specific uridine modifications in tRNA's wobble site. Moreover, this complex has been indirectly involved in the regulation of alpha-tubulin acetylation in microtubules (MTs) via the stabilization of ATP-Citrate Lyase (Acly), the main cytosolic source of acetyl-CoA production in cells, a key substrate used for global protein acetylation. Here, we report additional evidence that Elongator activity is important for proper cytoskeleton remodeling as cells lacking expression of Elp1 show morphology impairment; including distinct neurite process formation and disorganization and instability of MTs. Here, we show that loss of Elongator results in a reduction of expression of the microtubule associated protein Tau (MAPT). Tau, is a well-known key MT regulator in neurons whose lysines can be competitively acetylated or ubiquitylated. Therefore, we tested whether Tau is an indirect acetylation target of Elongator. We found that a reduction of Elongator activity leads to a decrease of lysine acetylation on Tau that favors its proteasomal degradation. This phenotype was prevented by using selective deacetylase or proteasomal inhibitors. Moreover, our data demonstrate that Acly's activity regulates the mechanism underlying Tau mediated neurite morphology defects found in Elp1 KD since both Tau levels and neurites morphology are restored due to Acly overexpression. This suggests a possible involvement of both Tau and Acly dysfunction in Familial Dysautonomia (FD), which is an autosomal recessive peripheral neuropathy caused by mutation in the ELP1 gene that severely affects Elp1 expression levels in the nervous system in FD patients in a similar way as found previously in Elp1 KD neuroblastoma cells. This work is supported by the Israel Science Foundation, ISF Grant No. 1688/16 and ISF Grant No. 1513/20. LN. is Research Director of the F.R.S- F.N.R.S. The work performed in the Nguyen laboratory is supported by ULiège (Crédit Classique), the F.R.S.-F.N.R.S. (PDR T.0185.20; EOS 0019118F-RG36), WELBIO (CR-2022A-12), the Fonds Leon Fredericq, the Fondation Médicale Reine Elisabeth, the Fondation Simone et Pierre Clerdent, the Belgian Science Policy (IAP-VII network P7/20), the ERANET Neuron (STEM-MCD and NeuroTalk), and the Win2Wal (ChipOmics; #2010126).

The axonal transport of vesicles promotes microtubule acetylation across species., Microtubules are polymerized dimers of α- and β-tubulin that underlie a broad range of cellular activities. Acetylation of α-tubulin by the acetyltransferase ATAT1 modulates microtubule dynamics and functions in neurons. However, it remains unclear how this enzyme acetylates microtubules over long distances in axons. Here, we show that loss of ATAT1 impairs axonal transport in neurons in vivo, and cell-free motility assays confirm a requirement of α-tubulin acetylation for proper bidirectional vesicular transport. Moreover, we demonstrate that the main cellular pool of ATAT1 is transported at the cytosolic side of neuronal vesicles that are moving along axons. Together, our data suggest that axonal transport of ATAT1-enriched vesicles is the predominant driver of α-tubulin acetylation in axons.

Summary: The protein p27Kip1 plays roles that extend beyond cell-cycle regulation during cerebral cortex development, such as the regulation of neuronal migration and neurite branching via signaling pathways that converge on the actin and microtubule cytoskeletons. Microtubule-dependent transport is essential for the maturation of neurons and the establishment of neuronal connectivity though synapse formation and maintenance. Here, we show that p27Kip1 controls the transport of vesicles and organelles along the axon of mice cortical projection neurons in vitro. Moreover, suppression of the p27Kip1 ortholog, dacapo, in Drosophila melanogaster disrupts axonal transport in vivo, leading to the reduction of locomotor activity in third instar larvae and adult flies. At the molecular level, p27Kip1 stabilizes the α-tubulin acetyltransferase 1, thereby promoting the acetylation of microtubules, a post-translational modification required for proper axonal transport. : Morelli et al. report that p27Kip1/Dacapo modulates the acetylation of microtubules in axons via stabilization of ATAT1, the main α-tubulin acetyltransferase. Its conditional loss leads to the reduction of bidirectional axonal transport of vesicles and mitochondria in vitro in mice and in vivo in Drosophila. Keywords: p27Kip1, dacapo, acetylation, axonal transport, ATAT1, alpha-tubulin, HDAC6, Drosophila, mouse, cerebral cortex

The protein p27(KiP1) plays roles that extend beyond cell-cycle regulation during cerebral cortex development, such as the regulation of neuronal migration and neurite branching via signaling pathways that converge on the actin and microtubule cytoskeletons. Microtubule-dependent transport is essential for the maturation of neurons and the establishment of neuronal connectivity though synapse formation and maintenance. Here, we show that p27(KiP1) controls the transport of vesicles and organelles along the axon of mice cortical projection neurons in vitro. Moreover, suppression of the p27(Kip1) ortho-log, dacapo, in Drosophila melanogaster disrupts axonal transport in vivo, leading to the reduction of locomotor activity in third instar larvae and adult flies. At the molecular level, p27(KiP1) stabilizes the alpha-tubulin acetyltransferase 1, thereby promoting the acetylation of microtubules, a post-translational modification required for proper axonal transport. We thank N. Krusy and C. D'Alessandro for technical assistance. We are grateful to M. Nachury, N. Perrimon, and P. Verstreken for sharing reagents. We thank S. Ormenese and S. Freeman for assistance at the GIGA imaging platform. We thank E. Pichinuk at the Blavatnik Center for Drug Discovery in Tel-Aviv University for facilities use and help. A.E. and M.W. are supported by the Israel Science Foundation (grant no. 8811688). L.N. is supported by the Fonds de la Recherche Scientifique-Fonds de la Recherche Scientifique (FRS-FNRS, PDR DevoCortex-T.007315), the Fonds Leon Fredericq, the Fondation Medicale Reine Elisabeth, the Fondation Simone et Pierre Clerdent, the Belgian Science Policy (IAP-VII network P7/20), the Actions de Recherche Concertee (ARC) (ARC11/16-01), and the European Research Area Network Neuron (ERA-NET) STEM-MCD.

Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegenerative disorder caused by degeneration of motor neurons. The cause for most cases of ALS is multi-factorial,this enhances the need to characterize and isolate specific biomarkers found in biological samples from ALS patients. To this end we use human mesenchymal stem cells (hMSC) derived from the bone marrow of six ALS patients (ALS hMSC) and identified two genes, Cytoplasmic FMR Interacting Protein 2 (CyFIP2) and Retinoblastoma (Rb) Binding Protein 9 (RbBP9) with a significant decrease in post transcriptional A to I RNA editing compared to hMSC of healthy individuals. At the transcriptional level we show abnormal expression of these two genes in ALS hMSC by quantitative real time-PCR (qRT-PCR) and Western blot suggesting a problem in the regulation of these genes in ALS. To strengthen this view we tested by qRT-PCR the expression of these genes in peripheral blood leukocytes (PBL) isolated from blood samples of 17 ALS patients and found that CyFIP2 and RbBP9 levels of expression were significantly different compared to the levels of expression of these two genes in 19 normal PBL samples. Altogether we found two novel ALS potential biomarkers in non-neural tissues from ALS patients that may have direct diagnostic and therapeutic implications to the disease.