Dysregulation of Ubiquitin-Proteasome System in Neurodegenerative Diseases

1Many neurodegenerative diseases are associated with aggregate-prone neurotoxic proteins (aggresomes or inclusion bodies) that perturb cellular homeostasis and neuronal function. These diseases are also categorically classed as proteinopathies, and comprise aggregation-prone components such as β-amyloid (Aβ) and tau in Alzheimer’s disease (AD), α-synuclein in Parkinson’s disease (PD), and polyglutamine (polyQ) repeat diseases.

2Alzheimer’s disease is the most common form of progressive neurodegeneration and represents the main cause of senile dementia affecting about 10% of the population over the age of 65 and about 50% of the population over 85. AD can be divided into two forms: familial AD and sporadic AD. Familial AD accounts for 5%∼10% of AD patients, where inherited autosomal dominant mutations in three genes have been considered as the primary causes of familial AD: these include mutations in the genes encoding β-amyloid precursor protein (APP) and presenilins (PS1 and PS2;;; ). In addition, sporadic AD is associated with polymorphisms in the Apolipoprotein E (APOE) gene, where the epsilon four allele is a strong genetic risk factor for late-onset AD. Accumulation and aggregation of neurotoxic proteins, such as β-amyloid (Aβ), hyperphosphorylated tau, ubiquitinated proteins, and other unfolded proteins in vulnerable brain regions such as the hippocampus and cortex in AD brain is central to disease pathogenesis. Two major pathological hallmarks in AD are extracellular amyloid plaques and intraneuronal neurofibrillary tangle (NFT; ). NFTs are comprised of hyperphosphorylated microtubule-associated protein tau. Amyloid plaques are primarily comprised of Aβ which is derived from sequential cleavages of APP by β- and γ-secretases. Cumulative evidence demonstrates that Aβ and tau are neurotoxic and can trigger a cascade of neurodegenerative processes ending in neuronal death, suggesting that overproduction/accumulation of Aβ and tau in vulnerable brain regions is the primary influence driving AD pathogenesis.

3Parkinson’s disease is the second most common neurodegenerative disease, clinically characterized by motor abnormalities, including resting tremor, rigidity, hypokinesia, and postural instability. The loss of dopaminergic neurons in the substantial nigra subregion of midbrain is the major cause of motor deficits. Major pathological features of PD include intraneuronal accumulation of Lewy bodies and dystrophic neurites (Lewy neurites), both of which comprise aggregated proteins, such as α-synuclein, parkin, and ubiquitinated proteins. Sporadic PD accounts for more than 90% of PD cases, and only less than 10% of familial PD cases are caused by monogenic mutations. Mutations in the gene encoding α-synuclein represent autosomal dominant familial PD, and mutations in genes encoding parkin, UCHL1, DJ-1, PINK1, and LRRK2 cause autosomal recessive familial PD.

4Huntington’s disease (HD) is an autosomal dominant progressive neurodegenerative disorder, clinically characterized by cognitive impairment, chorea and dystonia, bradykinesia, uncoordinated movement, mood disorder and other psychiatric symptoms, and behavioral difficulties. As the most common of the polyQ disorders, HD is caused by expansion of a CAG repeat coding for polyQ in the N-terminus of the huntingtin protein. There is a remarkable repeat length threshold effect, where polyQ chain expansion of 36 repeats triggers HD onset, whereas repeats of less than 35 are non-pathological. The polyQ expansion in the N-terminus of huntingtin protein confers a gain-of-function phenotype, and is associated with significant neurotoxicity.

5In addition to huntingtin, expansion of polyQ-encoding CAG repeats in several genes have also been found in other polyQ repeat diseases, such as Ataxin-1 in spinocerebellar ataxia type 1 (SCA1), Ataxin-2 in SCA2, Atantin-3 in SCA3, α1A-voltage-dependent calcium channel (CACNA1A) in SCA6, Ataxin-7 in SCA7, Atrophin-1 in dentatorubral-pallidoluysian atrophy (DRPLA), and androgen receptor in SBMA. All polyQ repeat disorders are dominant neurodegenerative diseases and are associated with pathological intracellular inclusion bodies in the affected brain regions.

6Ubiquitin is an evolutionarily conserved 76-amino acid moiety covalently tandemly linked to target protein components for degradation by the ubiquitin-proteasome system (UPS), and is required for degradation of about 80% intracellular proteins in eukaryotes. The presence of ubiquitin in intracellular inclusions has been found in various neurodegenerative diseases, for example, amyloid plaques and NFT in AD, Lewy bodies in PD, and intranuclear inclusions in polyQ disorders.

7Protein ubiquitination occurs through the coordinated activity of several enzymes, including an ubiquitin activating enzyme (E1 ligase), conjugating enzyme (E2 ligase), and E3 ligase. Initially, a single ubiquitin moiety is attached to an active-site Cysteine residue within the E1 ligase through a thioester bond in an ATP dependent manner. The activated ubiquitin is then transferred to an ubiquitin conjugating enzyme-E2 ligase. Finally, the E2 ligase will cooperatively transfer the ubiquitin chain with a specified E3 ligase to a particular substrate, whereby the 26S proteasome will target the polyubiquitinated protein for degradation (Figure 1).

8Ubiquitin E3 ligases determine the specificity of ubiquitination process. E3 ligases are grouped into three classes according to their unique domains, including Really Interesting New Gene (RING) or U-box domain-containing E3 ligases, Homologous to E6AP C-terminus (HECT) domain-containing E3 ligases, and RING-between-RING (RBR) domain-containing E3 ligases. RING E3 ligases catalyze the transfer of ubiquitin directly from E2 ligases to the substrates by binding both of them. Whereas HECT E3 ligases comprise a conserved Cysteine residue as an ubiquitin acceptor, where the activated ubiquitin is initially transferred from the E2 ligases to HECT E3 ligases prior to E3-mediated substrate conjugation. Unlike RING E3 ligases, RBR E3 ligases contain two RING domains (RING1 and RING2) separated by an in-between-RING (IBR) domain. Functionally, RBR E3 ligases are similar with HECT E3 ligases, both catalyze ubiquitin first transfer from E2 ligases to a catalytic Cysteine residue on E3 ligases then from the E3 ligases to the substrates (Figure 1).

9The fate of an ubiquitin-linked protein is dependent on Lysine chain-specific polyubiquitination. For example, Lys48-linked polyubiquitin chains usually target proteins for degradation, whereas Lys63-linked polyubiquitination is involved in regulation of NFκB signaling, Lys27-linked polyubiquitin chains are important for mitophagy, and Lys11-linked polyubiquitination is implicated in cell cycle regulation. Therefore, ubiquitination is not only important for protein degradation, but is also implicated in non-proteolytic functions, such as endocytosis, protein-protein interaction, intracellular trafficking, inflammatory signaling, autophagy, and DNA repair.

10The UPS is involved in protein quality control and removal of misfolded and aggregated proteins, and dysfunction of the UPS is implicated in the pathogenesis of neurodegenerative diseases.

11The process of ubiquitination is reversible and the reverse process is called deubiquitination which is accomplished by deubiquitinating enzymes (DUBs;;; ). Increasing evidence indicates the importance of DUBs in regulating UPS function, including proofreading ubiquitin-protein conjugates, removing ubiquitin from conjugated proteins as key examples (Figure 1). Pivotal roles for DUBs have been demonstrated in maintaining neuronal functions and regulating the pathogenesis of various neurodegenerative diseases.

12More than 100 DUBs have been identified, and can be subdivided into five subfamilies. DUBs can be further categorized into two classes, the Cysteine proteases and the zinc-dependent metalloproteases. Cysteine protease-associated DUBs include the ubiquitin carboxyl-terminal hydrolases (UCHs), ubiquitin specific proteases (USPs), Machado Joseph Diseases (MJD) proteases, and ovarian tumor (OTU) protease. Metalloprotease DUBs comprise the JAB1/MPN/Mov34 metalloenzyme (JAMM) subfamily.

13UCHL1, a member of the UCH subfamily, is one of the most abundant proteins in the brain. UCHL1 is known to interact with and stabilize monomeric ubiquitin, and facilitates the E3 ligase activity in its dimeric form. An Aplysia UCHL1 ortholog, ubiquitin carboxyl-terminal hydrolase (AP-Uch) has been found to be important in maintaining synaptic function. Inhibiting the expression or function of AP-Uch blocks induction of long-term facilitation, which is thought to be the molecular basis for learning and memory. Mammalian UCHL1 is required for normal synaptic structure and function in hippocampal neurons. Activation of NMDA receptor upregulates UCHL1 activity and the levels of free ubiquitin monomers. Inhibition of UCHL1 disrupts distribution of synaptic proteins, increases dendritic spine size and reduces spine density. However, restoration of ubiquitin in UCHL1-inhibited neurons rescues impaired synaptic structure. UCHL1 is also essential for normal structure and function of neuromuscular junctions (NMJs), reduction of synaptic vesicles and accumulation of tubulovesicular structures at the presynaptic nerve terminals have been observed in Uchl1 knockout mice. UCHL3, a homolog of UCHL1, is involved in spatial and working memory.

1419S proteasome-associated DUBs comprise three members: USP14, UCH37, and RPN11, which show differences in ubiquitin chains removals and consequent protein degradation. It has been reported that both USP14 and UCH37 prevent substrate degradation by removing ubiquitin chains and promoting proteasomal substrate dissociation. In contrast, RPN11 cleaves at the base of the ubiquitin-linked substrates and promotes substrate degradation. USP14 is essential for synaptic development and normal function of NMJs, which is mediated through ubiquitination and activation of c-Jun N-terminal kinase signaling. Pharmacological inhibition of USP14 enhances proteasomal degradation of several neurotoxic proteins, including tau, TDP14, and Ataxin-3. Usp14-deficient mice display motor neuron deficits and developmental NMJ defects. USP16, a chromosome 21-encoded gene, is involved in chromatin remodeling and cell cycle progression through regulated ubiquitination of histone H2A. USP16 interacts with HERC2 and modulates the ubiquitination in DNA repair machinery components. USP16 is upregulated in Down’s syndrome (DS) cells due to extrachromosomal triplication in trisomy 21, downregulation of USP16 partially restores the impaired proliferation in DS somatic stem cells. It has been reported that USP16 acts as a H2A DUB and regulates hematopoiesis and hematopoietic stem cell function. Moreover, USP16 is required for embryonic stem cell differentiation, where USP16 deficiency leads to embryonic lethality in mouse deletion models.

15The gene encoding USP25 also resides on human chromosome 21, USP25 is involved in protein degradation by the 26S proteasome. USP25 is also implicated in inflammation, and it has been found to facilitate removal of Lys63-linked ubiquitin chains from tumor necrosis factor-receptor associated factor 5/6 (TRAF5/6), Usp25-deficient mice show higher sensitivity to IL-17 dependent inflammation and autoimmunity.

16Accumulating evidence indicates that dysfunction of the UPS is a key factor to initiate and aggravate the pathogenesis of neurodegenerative diseases. We will focus on the dysregulation of ubiquitination and proteasomal degradation in neurodegenerative pathology in AD, PD, and HD (Table 1).

17The presence of ubiquitin in NFTs and amyloid plaques in AD brain has been described as early as 1987. Ubiquitin-targeted proteasomal activity declines with age Tg2576 AD mouse model brain, and Aβ treatment markedly attenuates proteasomal activity in cultured neurons. Aβ induced UPS dysfunction contributes to the accumulation of reticulon 3 (RTN3) in dystrophic neurites, and the presence of dystrophic neurites and clustering of tubular endoplasmic reticulum has been observed in RTN3 transgenic mouse brain. Interestingly, intracellular Aβ accumulation and impaired proteasome function can be reversed by the ubiquitin E3 ligase parkin. Parkin expression is downregulated in AD brains, and reduced parkin expression may contribute to the accumulation of intracellular Aβ. Moreover, parkin expression results in Aβ reduction which can be dampened by proteasome inhibitors. In addition, overexpression of parkin in APP/PS1 mouse models restores impaired long-term potentiation (LTP) and rescues behavioral abnormalities by reducing Aβ load and neuroinflammation.

18UCHL1 has been observed in ubiquitin-enriched inclusion bodies in AD brains. In addition, decreased soluble UCHL1 protein levels was detected in the brains of postmortem AD patients and APP/PS1 mouse models, and soluble UCHL1 is inversely proportional to the number of NFTs in AD brains. Upregulation of UCHL1 improves contextual learning and restores synaptic functions in APP/PS1 mouse models, where rescue effects are dependent on enzymatic UCHL1 activity. Moreover, overexpression of UCHL1 reduces the levels of β-secretase BACE1, and consequent BACE1 cleavage products (APP C-terminal fragment C99 and Aβ). Depletion of UCHL1 increases the levels of BACE1, C99, and Aβ in the Uchl1-null gad mice.

19A direct interaction between APP and ubiquitin ligases also contributes to amyloid pathology. HRD1, an endoplasmic reticulum-associated degradation (ERAD) ubiquitin E3 ligase, is reduced in AD brains. HRD1 interacts with APP through its Proline-rich region, and this interaction reduces Aβ generation through promoting APP ubiquitination and degradation. In addition, APP can bind to ubiquitin E3 ligases Stub1 and CRL4CRBN through the APP cytosolic region (ACR; ). ACR promotes the ubiquitination of several presynaptic proteins and regulates neurotransmitter release.

20Polyubiquitinated tau has also been found in the brains of AD patients, where ubiquitin conjugation usually occurs at Lys254, Lys311, and Lys353 within the tau microtubule-binding domain. In AD brains, accumulation of hyperphosphorylated, ubiquitinated tau protein has been found at both presynaptic and postsynaptic terminals, which is associated with dysfunction of the UPS. Recently, Myeku et al. reported that tau can induce dysfunction of the 26S proteasome which can be prevented by activating cAMP-PKA signaling. Protective effects derived from cAMP-PKA signaling are mediated through phosphorylation of the 26S proteasomal subunit. Han et al. directly delivered purified proteasomes into cells through mesoporous silica nanoparticle-mediated endocytosis, and they found that extraproteasomal transfer reduces tau aggregates and promotes cell survival against tau-induced proteotoxic stress. C-terminus of Hsp70-interacting protein (CHIP), an ubiquitin E3 ligase, is also implicated in AD pathogenesis. It has been found that both CHIP and Hsp70 are upregulated in the brains of AD patients. CHIP and its binding protein Hsp70 can induce the ubiquitination and degradation of phosphorylated tau, deletion of CHIP leads to the accumulation of non-aggregated, hyperphosphorylated, as well as caspase-3-cleaved tau. Moreover, inhibition of Hsp90 reduces hyperphosphorylated tau through an interaction with CHIP. In addition, overexpression of CHIP and Hsp70/90 decreases steady-state Aβ levels and promotes Aβ degradation.

21The presence of ubiquitin-immunopositive Lewy bodies in the brains of PD patients has been observed about 30 years ago. Impaired proteasomal function in the substantia nigra of PD brains was later observed. Importantly, genetic evidence reveals a clear involvement of UPS dysfunction in PD pathogenesis: familial PD-associated genetic variations have been identified on PARK2 and UCHL1, which encode the ubiquitin E3 ligase parkin and deubiquitinating enzyme UCHL1, respectively. Recessive early-onset familial PD can also be caused by mutations in genes encoding DJ-1 and PINK1, presumably through a loss-of-function mechanism.

22An interaction between parkin and its substrate α-synuclein is a crucial regulatory component of dopaminergic neuron degeneration, loss-of-function mutations in PARK2 usually cause early-onset autosomal recessive PD, whereas disease-associated mutations in α-synuclein are usually associated with autosomal dominant PD. Mutant parkin fails to interact with α-synuclein, leads to the accumulation of α-synuclein and the formation of Lewy bodies. Transgenic mice carrying mutant parkinQ311X develop progressive hypokinetic motor deficits, accumulation of α-synuclein and age-dependent loss of dopaminergic neurons in the substantia nigra and striatum.

23Parkin is an E3 ligase mediating multiple forms of ubiquitination, including monoubiquitination, Lys48- and Lys63-linked polyubiquitination. In addition, parkin can form an E3 ligase complex with PINK1 and DJ-1 to promote unfolded protein degradation: PD mutations in each component impair E3 ligase activity. CHIP can promote parkin-mediated ubiquitination of Pael receptor and inhibit the Pael receptor-induced cell death. In addition, CHIP ubiquitinates and facilitates proteasomal degradation of LRRK2, where knockdown of CHIP exacerbates neurotoxicity mediated by mutant LRRK2. CHIP can ubiquitinate α-synuclein and reduce toxic α-synuclein oligomers. TRAF6 is upregulated in the brains of PD patients, and TRAF6 promotes Lys6-, Lys27-, and Lys29-linked ubiquitination of DJ-1 and α-synuclein. This interaction may cause aggregation of insoluble and polyubiquitinated mutant DJ-1 proteins. Moreover, TRAF6 ubiquitinates PINK1 at Lys433 residue as a poly-Lys63 conjugated form which stabilizes PINK1 in depolarized mitochondria. Down-regulation of TRAF6 disrupts PINK1 localization, thereby causing mitochondria damage through parkin recruitment. Mutations in UCHL1 have been reported in rare cases of early-onset familial PD. Most notably, the UCHL1I93M PD variant shows reduced deubiquitinating activity. Ubiquitin-positive inclusion bodies and the loss of dopaminergic neurons have been observed in the substantia nigra of UCHLI93M transgenic mouse brains. In addition, UCHL1I93M mutation promotes dimerization and Lys63-linked polyubiquitination of α-synuclein. Lys63-linked polyubiquitination may aggravate PD pathology by inhibiting proteasomal degradation of α-synuclein.

24HD patients manifest polyQ repeats of over 40 residues within the N-terminus of huntingtin protein. Ubiquitin-linked mutant huntingtin has been found in neuronal intranuclear inclusions and dystrophic neurites in the cortex and striatum of HD patients. However, it seems that eukaryotic proteasomes fail to cleave and eliminate pathogenic protein species with repeats greater than 25Q residues; this results in accumulation and aggregation of huntingtin protein in the neuronal nucleus. However, it has been reported that a selective histone deacetylase (HDAC) inhibitor 4b ameliorates cognitive impairment in N171-82Q HD mouse model by preventing formation of nuclear huntingtin aggregates. Mechanistically, HDAC inhibition upregulates expression of multiple genes that are fundamental to protein phosphorylation and ubiquitination, including Ube2K, Ubqln, Ube2e3, Usp28, Sumo2, as well as the genes encoding components of the inhibitor of κB kinase (IKK) complex. It has been reported that CHIP suppresses polyQ aggregation and toxicity, and knockdown of CHIP in HD transgenic mice aggravated disease pathology.

25Spinocerebellar ataxia type 3 (SCA3) is another polyQ repeat disease, which is caused by a polyQ-encoding CAG repeat expansion in the gene encoding Ataxin-3. SCA3 is characterized by progressive ataxia with degeneration in the cerebellum, brain stem, substantia nigra, and globus pallidus interna. Clinical symptoms of SCA3 include impaired eye movement, speech and swallowing defects. Ataxin-3 is a DUB from MJD subfamily of proteases which contain an ubiquitin interacting motif flanking the polyQ domain. Ataxin-3 preferentially cleaves Lys63 linkages in polyubiquitinated chains containing both Lys48 and Lys63 linkages. Overexpression of normal human Ataxin-3 can protect Drosophila from polyQ-huntingtin-induced neurodegeneration. Unlike wild-type Ataxin-3, MJD-linked mutant Ataxin-3 can promote ubiquitination and autophagic degradation of parkin probably through regulating the levels of Lys27- and Lys29 ubiquitin-linked parkin.

26Although UPS-mediated protein degradation has been well-studied in the last few decades, regulation of ubiquitination and proteasomal degradation in normal brain function and pathogenesis of neurodegenerative diseases remains largely unknown. The regulation of polyubiquitination processes may vary at different physiological and/or pathologic conditions. This review surveyed dysregulation of UPS in several major neurodegenerative diseases, more research is required to understand how the UPS is disrupted in neurodegenerative pathogenesis. By enriching our understanding of protein degradation, we can begin to formulate strategies to treat neurodegenerative diseases remediating aspects of UPS dysregulation.

27QZ, LZ, and XW wrote the manuscript, TH, YZ, HL, and HX edited the manuscript. All authors read and approved the final manuscript.

28The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.