KIF5A is a neuron-specific kinesin involved in anterograde axonal transport. Heterozygous missense mutations targeting KIF5A motor or stalk domain determine the development of hereditary spastic paraplegia (HSP) or Charcot-Marie-Tooth disease type 2 (CMT2), while de novo frameshift mutations in its cargo domain are associated to amyotrophic lateral sclerosis (ALS) and neonatal intractable myoclonus (NEIMY). The molecular mechanisms underlying KIF5A-dependent pathogenesis are not known yet, neither is the reason why mutations in different KIF5A domains cause distinct phenotypes. Therefore, we aim to investigate which processes are at the basis of KIF5A-associated disorders.We performed genetic testing on a large cohort of Italian patients affected by neuromuscular or neurodevelopmental disorders and selected seven KIF5A mutations spanning all the three domains of the corresponding protein and associated to one of the four above-mentioned phenotypes. Four mutations were already present in the literature (R280C, R864*, P986L, N999Vfs*73), while three are novel. We overexpressed the selected KIF5A mutants in SH-SY5Y neuroblastoma cells for their functional characterization, in comparison to cells overexpressing wild-type KIF5A. Firstly, we observed reduced protein levels for two novel KIF5A mutants that we could attribute to enhanced protein turnover. Secondly, we studied whether the autophagic flux is altered in our models, since autophagosomes are known to be transported by conventional kinesins and autophagy impairments are associated to several neuromuscular diseases – including ALS. We detected no significant differences in SQSTM1, MAP1LC3, and TFEB mRNA levels and in p62, LC3, and HSP70 protein levels between all tested conditions. Moreover, minimal alterations in the early response to proteotoxic stress were evidenced in our models upon sodium arsenite administration. Collectively, our preliminary data show that autophagy is not impaired upon mutant KIF5A overexpression in SH-SY5Y cells in basal and stress conditions, even in the presence of unstable KIF5A variants that might tamper with such degradative process.

Analysis of the autophagic response in models of KIF5A-related neurodegeneration / M. Cozzi, B. Tedesco, R. Cristofani, M. Galbiati, P. Rusmini, V. Crippa, M. Piccolella, V. Ferrari, E. Casarotto, M. Chierichetti, F. Mina, C. Gellera, S. Magri, F. Taroni, A. Poletti. ((Intervento presentato al 1. convegno ESN Virtual Conference : Future perspectives for European neurochemistry – a young scientists conference tenutosi a online nel 2021.

Analysis of the autophagic response in models of KIF5A-related neurodegeneration

M. Cozzi;B. Tedesco;R. Cristofani;M. Galbiati;P. Rusmini;V. Crippa;M. Piccolella;V. Ferrari;E. Casarotto;M. Chierichetti;F. Mina;S. Magri;A. Poletti
2021-05-26

Abstract

KIF5A is a neuron-specific kinesin involved in anterograde axonal transport. Heterozygous missense mutations targeting KIF5A motor or stalk domain determine the development of hereditary spastic paraplegia (HSP) or Charcot-Marie-Tooth disease type 2 (CMT2), while de novo frameshift mutations in its cargo domain are associated to amyotrophic lateral sclerosis (ALS) and neonatal intractable myoclonus (NEIMY). The molecular mechanisms underlying KIF5A-dependent pathogenesis are not known yet, neither is the reason why mutations in different KIF5A domains cause distinct phenotypes. Therefore, we aim to investigate which processes are at the basis of KIF5A-associated disorders.We performed genetic testing on a large cohort of Italian patients affected by neuromuscular or neurodevelopmental disorders and selected seven KIF5A mutations spanning all the three domains of the corresponding protein and associated to one of the four above-mentioned phenotypes. Four mutations were already present in the literature (R280C, R864*, P986L, N999Vfs*73), while three are novel. We overexpressed the selected KIF5A mutants in SH-SY5Y neuroblastoma cells for their functional characterization, in comparison to cells overexpressing wild-type KIF5A. Firstly, we observed reduced protein levels for two novel KIF5A mutants that we could attribute to enhanced protein turnover. Secondly, we studied whether the autophagic flux is altered in our models, since autophagosomes are known to be transported by conventional kinesins and autophagy impairments are associated to several neuromuscular diseases – including ALS. We detected no significant differences in SQSTM1, MAP1LC3, and TFEB mRNA levels and in p62, LC3, and HSP70 protein levels between all tested conditions. Moreover, minimal alterations in the early response to proteotoxic stress were evidenced in our models upon sodium arsenite administration. Collectively, our preliminary data show that autophagy is not impaired upon mutant KIF5A overexpression in SH-SY5Y cells in basal and stress conditions, even in the presence of unstable KIF5A variants that might tamper with such degradative process.
Settore BIO/13 - Biologia Applicata
European Society for Neurochemistry-Young Scientist Steering Committee
Analysis of the autophagic response in models of KIF5A-related neurodegeneration / M. Cozzi, B. Tedesco, R. Cristofani, M. Galbiati, P. Rusmini, V. Crippa, M. Piccolella, V. Ferrari, E. Casarotto, M. Chierichetti, F. Mina, C. Gellera, S. Magri, F. Taroni, A. Poletti. ((Intervento presentato al 1. convegno ESN Virtual Conference : Future perspectives for European neurochemistry – a young scientists conference tenutosi a online nel 2021.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/847026
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