Rab proteins compose the largest family of small GTPases and control the different steps of intracellular membrane traffic. More recently, they have been shown to also regulate cell signaling, division, survival, and migration. The regulation of these processes generally occurs through recruitment of effectors and regulatory proteins, which control the association of Rab proteins to membranes and their activation state. Alterations in Rab proteins and their effectors are associated with multiple human diseases, including neurodegeneration, cancer, and infections. This review provides an overview of how the dysregulation of Rab-mediated functions and membrane trafficking contributes to these disorders. Understanding the altered dynamics of Rabs and intracellular transport defects might thus shed new light on potential therapeutic strategies.

The intracellular movement and positioning of organelles and vesicles is mediated by the cytoskeleton and molecular motors. Small GTPases like Rab and Arf proteins are main regulators of intracellular transport by connecting membranes to cytoskeleton motors or adaptors. However, it is becoming clear that interactions between these small GTPases and the cytoskeleton are important not only for the regulation of membrane transport. In this review, we will cover our current understanding of the mechanisms underlying the connection between Rab and Arf GTPases and the cytoskeleton, with special emphasis on the double role of these interactions, not only in membrane trafficking but also in membrane and cytoskeleton remodeling. Furthermore, we will highlight the most recent findings about the fine control mechanisms of crosstalk between different members of Rab, Arf, and Rho families of small GTPases in the regulation of cytoskeleton organization.

The serpinopathies are human pathologies caused by mutations that promote polymerisation and intracellular deposition of proteins of the serpin superfamily, leading to a poorly understood cell toxicity. The dementia FENIB is caused by polymerisation of the neuronal serpin neuroserpin (NS) within the endoplasmic reticulum (ER) of neurons. With the aim of understanding the toxicity due to intracellular accumulation of neuroserpin polymers, we have generated transgenic neural progenitor cell (NPC) cultures from mouse foetal cerebral cortex, stably expressing the control protein GFP (green fluorescent protein), or human wild type, G392E or delta NS. We have characterised these cell lines in the proliferative state and after differentiation to neurons. Our results show that G392E NS formed polymers that were mostly retained within the ER, while wild type NS was correctly secreted as a monomeric protein into the culture medium. Delta NS was absent at steady state due to its rapid degradation, but it was easily detected upon proteasomal block. Looking at their intracellular distribution, wild type NS was found in partial co-localisation with ER and Golgi markers, while G392E NS was localised within the ER only. Furthermore, polymers of NS were detected by ELISA and immunofluorescence in neurons expressing the mutant but not the wild type protein. We used control GFP and G392E NPCs differentiated to neurons to investigate which cellular pathways were modulated by intracellular polymers by performing RNA sequencing. We identified 747 genes with a significant upregulation (623) or downregulation (124) in G392E NS-expressing cells, and we focused our attention on several genes involved in the defence against oxidative stress that were up-regulated in cells expressing G392E NS (Aldh1b1, Apoe, Gpx1, Gstm1, Prdx6, Scara3, Sod2). Inhibition of intracellular anti-oxidants by specific pharmacological reagents uncovered the damaging effects of NS polymers. Our results support a role for oxidative stress in the cellular toxicity underlying the neurodegenerative dementia FENIB.

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The neuronal serpin neuroserpin undergoes polymerisation as a consequence of point mutations that alter its conformational stability, leading to a neurodegenerative dementia called familial encephalopathy with neuroserpin inclusion bodies (FENIB). Neuroserpin is a glycoprotein with predicted glycosylation sites at asparagines 157, 321 and 401. We used site‐directed mutagenesis, transient transfection, western blot, metabolic labelling and ELISA to probe the relationship between glycosylation, folding, polymerisation and degradation of neuroserpin in validated cell models of health and disease. Our data show that glycosylation at N157 and N321 plays an important role in maintaining the monomeric state of neuroserpin, and we propose this is the result of steric hindrance or effects on local conformational dynamics that can contribute to polymerisation. Asparagine residue 401 is not glycosylated in wild type neuroserpin and in several polymerogenic variants that cause FENIB, but partial glycosylation was observed in the G392E mutant of neuroserpin that causes severe, early‐onset dementia. Our findings indicate that N401 glycosylation reports lability of the C‐terminal end of neuroserpin in its native state. This C‐terminal lability is not required for neuroserpin polymerisation in the endoplasmic reticulum, but the additional glycan facilitates degradation of the mutant protein during proteasomal impairment. In summary, our results indicate how normal and variant‐specific N‐linked glycosylation events relate to intracellular folding, misfolding, degradation and polymerisation of neuroserpin.