Ubiquitin signalling in neurodegeneration: mechanisms and therapeutic opportunities

Abstract

Neurodegenerative diseases are characterised by progressive damage to the nervous system including the selective loss of vulnerable populations of neurons leading to motor symptoms and cognitive decline. Despite millions of people being affected worldwide, there are still no drugs that block the neurodegenerative process to stop or slow disease progression. Neuronal death in these diseases is often linked to the misfolded proteins that aggregate within the brain (proteinopathies) as a result of disease-related gene mutations or abnormal protein homoeostasis. There are two major degradation pathways to rid a cell of unwanted or misfolded proteins to prevent their accumulation and to maintain the health of a cell: the ubiquitin-proteasome system and the autophagy-lysosomal pathway. Both of these degradative pathways depend on the modification of targets with ubiquitin. Aging is the primary risk factor of most neurodegenerative diseases including Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. With aging there is a general reduction in proteasomal degradation and autophagy, and a consequent increase of potentially neurotoxic protein aggregates of β-amyloid, tau, α-synuclein, SOD1 and TDP-43. An often over-looked yet major component of these aggregates is ubiquitin, implicating these protein aggregates as either an adaptive response to toxic misfolded proteins or as evidence of dysregulated ubiquitin-mediated degradation driving toxic aggregation. In addition, non-degradative ubiquitin signalling is critical for homoeostatic mechanisms fundamental for neuronal function and survival, including mitochondrial homoeostasis, receptor trafficking and DNA damage responses, whilst also playing a role in inflammatory processes. This review will discuss the current understanding of the role of ubiquitin-dependent processes in the progressive loss of neurons and the emergence of ubiquitin signalling as a target for the development of much needed new drugs to treat neurodegenerative disease.

Fig. 1. Ubiquitin in degradation and cell signalling.

A Schematic of the ubiquitination cascade and opposing deubiquitination. During ubiquitination, ubiquitin is first attached to an E1 ubiquitin activating enzyme in an ATP-dependent manner before being transferred to an E2 ubiquitin conjugating enzyme. The Ub~E2 conjugate is then recognised by an E3 ubiquitin ligase, and ubiquitin is then transferred either directly to a substrate (for RING family E3 ubiquitin ligases) or first attached to the E3 ubiquitin ligase before being transferred to the substrate (for HECT and RBR family E3 ubiquitin ligases). The reverse reaction, deubiquitination, is catalysed by the deubiquitinating enzymes (DUBs) which remove ubiquitin from ubiquitinated substrates. B Ubiquitin–proteasome system. Ubiquitination of substrates can target proteins directly, or via ubiquitin-binding shuttle proteins such as UBQLN2, to the proteasome for proteolysis. Deubiquitination at the proteasome by proteasome-associated DUBs (USP14, UCH37) can rescue substrates from degradation. C Autophagy–lysosomal pathway. Specific E3 ligases ubiquitinate protein aggregates (e.g. CHIP, NEDD4, TRAF6), or damaged organelles (e.g. Parkin), to recruit autophagy receptors (e.g. p62, Neighbour of BRCA1 gene 1 (NBR1), OPTN, NDP52, TAX1BP1) that can simultaneously bind ubiquitin and Atg8-like proteins (LC3s). The Atg proteins catalyse the conjugation of the lipid phosphatidylethanolamine (PE) to LC3s and mediate the expansion of the autophagophore. D Non-degradative ubiquitin signalling. Ubiquitination can influence multiple pathways through degradative mechanisms or through non-degradative mechanisms. Non-proteolytic roles of ubiquitin often involve atypical chain linkage types.

Fig. 2. Ubiquitin signalling in the neurodegenerative brain.

Degradation-dependent and degradation-independent ubiquitin signalling plays a fundamental role in neuronal functioning and survival and disrupted control of these processes can trigger neuronal loss. Defective clearance of misfolded and ubiquitinated protein aggregates (such as Aβ, tau in AD; α-synuclein in PD; htt in HD; SOD1, TDP-43 in ALS) due to impaired proteasomal degradation and/or impaired autophagic–lysosomal degradation are a hallmark of neurodegenerative disease. Disrupted mitophagy: The pathological effect of defective mitochondria is exacerbated by impaired ubiquitin-mediated mitophagy due to compromised PINK1 or the E3 ubiquitin ligase Parkin. Damaged mitochondria are a potent source of DAMPS (e.g. mtDNA) and damaging reactive oxygen species. Dysregulated inflammation: Toxic protein aggregates, mtDNA and myelin deposits trigger inflammasome formation and the release of pro-inflammatory cytokines such as IL-1β and IL-18 and other neurotoxic factors by reactive astrocytes and microglia. Increased activity of the immunoproteasome in microglia further drives an inflammatory response contributing to disease pathology. Impaired DNA damage response: Disrupted ubiquitin signalling in the DNA damage response (e.g. via perturbed ubiquitination of histones). Dysregulated receptor trafficking: Endolysosomal trafficking such as that mediated by NEDD4 regulates cell surface expression of key neuronal receptors including EGF and AMPA receptors.

Fig. 3. Parkin-mediated mitophagy.

Chronic stresses of aging such as ROS and accumulated mitochondrial DNA (mtDNA) mutations provoke mitochondrial defects and damage that need to be counteracted by mitophagy to maintain neuronal cell survival. Upon mitochondrial damage, PINK1 is stabilised on the mitochondrial outer membrane where it autophosphorylates and activates (inset A). Active PINK1 phosphorylates ubiquitin, which recruits cytosolic Parkin to mitochondria. PINK1 then phosphorylates and activates phospho-ubiquitin-bound Parkin, enabling Parkin to ubiquitinate proteins on the mitochondrial outer membrane. Deubiquitinases, including USP30, remove ubiquitin from mitochondrial outer membrane proteins either upstream or downstream of Parkin activity to limit mitophagy (inset B). Mitochondrial ubiquitination leads to the recruitment of autophagy receptor proteins such as NDP52 and OPTN that is promoted by phosphorylation by TANK Binding Kinase 1 (TBK1), and the subsequent recruitment of the Unc-51 like autophagy activating kinase (ULK1) complex drives autophagophore biogenesis and encapsulation of mitochondria (inset C). Subsequent autophagosome-lysosome fusion leads to mitochondrial degradation by lysosomal hydrolases.

Fig. 4. Ubiquitin signalling as a target to treat neurodegenerative disease.

Defective ubiquitin signalling is an Achilles heel of neurons in neurodegenerative disease. Pre-clinical drug development to enhance neuroprotective (green arrow) or to inhibit neurodegenerative (red block) activity of targets, include stimulating protein quality control through modulating the deubiquitinating enzymes USP14 or UCHL1 (A), molecules that stimulate mitophagy by activating PINK1 or Parkin, or by inhibiting USP30 (B), and targeted degradation technologies (PROTACs and AUTACs) that recruit the ubiquitination machinery to induce selective ubiquitination and degradation of a target through either UPS or autophagy (C). Inhibitors of UCHL1 may also provide protection by stimulating aggrephagy. Compound structures were generated using ChemDraw 19.1.