Deubiquitylating enzymes (DUBs), act downstream of ubiquitylation. As such, these post-post-translational modifiers function as the final arbitrators of a protein substrate’s ubiquitylation status, thus regulating its fate. In most instances, DUBs moderate the absolute level of a substrate, its locality or activity, rather than being an “all-or-none” phenomenon. Yet, disruption of this quantitative regulation can produce dramatic qualitative differences. The ubiquitin-specific protease 9X (USP9X/FAM) is a substrate-specific DUB, which displays an extraordinarily high level of sequence conservation from Drosophila to mammals. It is primarily the recent revelations of USP9X’s pivotal role in human cancers, both as oncogene or tumour suppressor, in developmental disorders including intellectual disability, epilepsy, autism and developmental delay that has led to a subsequent re-examination of its molecular and cellular functions. Results from experimental animal models have implicated USP9X in neurodegeneration, including Parkinson’s and Alzheimer’s disease, as well as autoimmune diseases. In this review, we describe the current and accumulated knowledge on the molecular, cellular and developmental aspects of USP9X function within the context of the biological consequences during normal development and disease.

In humans, the Crumbs homolog-1 (CRB1) gene is mutated in progressive types of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. However, there is no clear genotype-phenotype correlation for CRB1 mutations, which suggests that other components of the CRB complex may influence the severity of retinal disease. Therefore, to understand the physiological role of the Crumbs complex proteins, we generated and analysed conditional knockout mice lacking CRB2 in the developing retina. Progressive disorganization was detected during late retinal development. Progressive thinning of the photoreceptor layer and sites of cellular mislocalization was detected throughout the CRB2-deficient retina by confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. Under scotopic conditions using electroretinography, the attenuation of the a-wave was relatively stronger than that of the b-wave, suggesting progressive degeneration of photoreceptors in adult animals. Histological analysis of newborn mice showed abnormal lamination of immature rod photoreceptors and disruption of adherens junctions between photoreceptors, Müller glia and progenitor cells. The number of late-born progenitor cells, rod photoreceptors and Müller glia cells was increased, concomitant with programmed cell death of rod photoreceptors. The data suggest an essential role for CRB2 in proper lamination of the photoreceptor layer and suppression of proliferation of late-born retinal progenitor cells.

Olfactory ensheathing cells (OECs) are glia reported to sustain the continuous axon extension and successful topographic targeting of the olfactory receptor neurons responsible for the sense of smell (olfaction). Due to this distinctive property, OECs have been trialed in human cell transplant therapies to assist in the repair of central nervous system injuries, particularly those of the spinal cord. Though many studies have reported neurological improvement, the therapy remains inconsistent and requires further improvement. Much of this variability stems from differing olfactory cell populations prior to transplantation into the injury site. While some studies have used purified cells, others have used unpurified transplants. Although both preparations have merits and faults, the latter increases the variability between transplants received by recipients. Without a robust purification procedure in OEC transplantation therapies, the full potential of OECs for spinal cord injury may not be realised.

The central nervous system (CNS) has very limited capacity to regenerate after traumatic injury or disease. In contrast, the peripheral nervous system (PNS) has far greater capacity for regeneration. This difference can be partly attributed to variances in glial-mediated functions, such as axon guidance, structural support, secretion of growth factors and phagocytic activity. Due to their growth-promoting characteristic, transplantation of PNS glia has been trialed for neural repair. After peripheral nerve injuries, Schwann cells (SCs, the main PNS glia) phagocytose myelin debris and attract macrophages to the injury site to aid in debris clearance. One peripheral nerve, the olfactory nerve, is unique in that it continuously regenerates throughout life. The olfactory nerve glia, olfactory ensheathing cells (OECs), are the primary phagocytes within this nerve, continuously clearing axonal debris arising from the normal regeneration of the nerve and after injury. In contrast to SCs, OECs do not appear to attract macrophages. SCs and OECs also respond to and phagocytose bacteria, a function likely critical for tackling microbial invasion of the CNS via peripheral nerves. However, phagocytosis is not always effective; inflammation, aging and/or genetic factors may contribute to compromised phagocytic activity. Here, we highlight the diverse roles of SCs and OECs with the focus on their phagocytic activity under physiological and pathological conditions. We also explore why understanding the contribution of peripheral glia phagocytosis may provide us with translational strategies for achieving axonal regeneration of the injured nervous system and potentially for the treatment of certain neurological diseases.

USP9X, is highly expressed in neural progenitors and, essential for neural development in mice. In humans, mutations in USP9X are associated with neurodevelopmental disorders. To understand USP9X’s role in neural progenitors, we studied the effects of altering its expression in both the human neural progenitor cell line, ReNcell VM, as well as neural stem and progenitor cells derived from Nestin-cre conditionally deleted Usp9x mice. Decreasing USP9X resulted in ReNcell VM cells arresting in G0 cell cycle phase, with a concomitant decrease in mTORC1 signalling, a major regulator of G0/G1 cell cycle progression. Decreased mTORC1 signalling was also observed in Usp9x-null neurospheres and embryonic mouse brains. Further analyses revealed, (i) the canonical mTORC1 protein, RAPTOR, physically associates with Usp9x in embryonic brains, (ii) RAPTOR protein level is directly proportional to USP9X, in both loss- and gain-of-function experiments in cultured cells and, (iii) USP9X deubiquitlyating activity opposes the proteasomal degradation of RAPTOR. EdU incorporation assays confirmed Usp9x maintains the proliferation of neural progenitors similar to Raptor-null and rapamycin-treated neurospheres. Interestingly, loss of Usp9x increased the number of sphere-forming cells consistent with enhanced neural stem cell self-renewal. To our knowledge, USP9X is the first deubiquitylating enzyme shown to stabilize RAPTOR.