Parkinson disease (PD) is one of the most well-studied neurological diseases related to E3 ligase dysfunction. PD is characterized by dystonia, rigidity, tremors, hyperreflexia, bradykinesia, postural instability, substantia nigra gliosis and dopamine depletion, and Lewy body dementia (Halliday et al., 2014; Biundo et al., 2016). The prevalence of this disease occurs in 0.3% of the general population in the United States and 0.1–0.2% in European countries with increasing rates that occur with aging (Kowal et al., 2013; Tysnes and Storstein, 2017). Genetic mutations in the E3 ligases LRSAM1, FBXO7 (PARK 15), and PARK2 (Figure 1 and Supplementary Table 1) have been identified in Parkinson disease (Wu et al., 2005; Choi et al., 2008; Lohmann et al., 2015; Aerts et al., 2016). PARKIN/PARK2, belongs to the RBR family of E3 ligases, and mutations in this gene occurs in 50% of familial cases and 10–20% in sporadic cases with high penetrance in early-onset PD (Lill, 2016; Zhang et al., 2016).

A series of advanced technologies have elucidated the functional consequences of deletion and missense mutations in the PARKIN gene (Wu et al., 2005; Choi et al., 2008) and their use has increased the capacity for PD therapeutics (Hattori et al., 1998; Kitada et al., 1998; Hedrich et al., 2001). For example, proteomic profiling demonstrated PARKIN's role in mitochondrial autophagy (Narendra and Youle, 2011). These findings were further supported by liquid chromatography–mass spectrometry and ubiquitin Absolute Quantification of ubiquitin (UB-AQUA) proteomics. UB-AQUA is a mass spectrometry-based method that uses internal standard peptides that are isotopically labeled to quantify peptides from digested mono- and poly-ubiquitinated chains attached on substrates (Kirkpatrick et al., 2006; Phu et al., 2011). UB-AQUA was used to identify and quantify subtypes of mitochondrial ubiquitin chain linkages. Using this method, PTEN-induced putative kinase 1 (PINK1) was found to phosphorylate PARKIN leading to its activation and formation of canonical and non-canonical ubiquitin chains on mitochondria, which were PARKIN-dependent (Ordureau et al., 2014). This served as a feed-forward mechanism to promote PARKIN recruitment and mitochondrial ubiquitination in response to mitochondrial damage. Collectively, these findings were critical to understanding potential causative factors leading to PD pathogenesis (Ordureau et al., 2014).

The impairment of mitochondria in PD due to deletions of mitochondrial DNA (mtDNA) leads to respiratory-chain deficiencies especially in dopamine (DA) neurons of the substantia nigra (Bender et al., 2006). To study the effects of mtDNA deletions and other genetic mutations, conditional knock-out or knock-in mouse models using CRE recombinase and loxP sites were used (Soriano, 1999). The CRE-loxP system allows for conditional loss-of-function or gain-of-function studies in specific tissues and circumvents early life lethality and unwanted phenotypes in later stages of life by controlling when genes are expressed temporally and spatially (Sauer, 1998). A MitoPark reporter mouse line was created first by inserting a loxP-flanked stop-cassette upstream of the mitochondrial targeting presequence lox and yellow fluorescent protein (YFP) transgene to target it to the mitochondrial matrix. The presence of the stop cassette limits YFP expression in specific cells and is only expressed when the stop cassette is excised out with CRE.

Using the MitoPark mouse model, the consequences of respiratory chain dysfunction on the properties of mitochondria and DA neurons were examined after DA neuron-specific knockout of the mitochondrial transcription factor A (TFAM) (Sterky et al., 2011). In order to study the DA neurons, ROSA26^+/SmY mice were crossed with dopamine transporter (DAT)-CRE mice that express CRE under a DA transporter locus, so the offspring from these parents express YFP precisely in the mitochondria of DA neurons in the midbrain (Sterky et al., 2011). Using this model, Sterky et al. demonstrated increased fragmented and aggregated mitochondria in aged PARKIN knockout mice (Sterky et al., 2011). Specifically, striatal DA neurons displayed a reduction in mitochondria and tyrosine hydroxylase density. Surprisingly, PARKIN knockout MitoPark mice presented no difference in morphology or number of mitochondria with or without PARKIN. There was also no indication of PARKIN recruitment to defective mitochondria suggesting PARKIN did not have an effect on the progression of neurodegeneration in PD (Sterky et al., 2011).

Although limitations exist in using the PD mouse model, there have been advancements in studying the role of PARKIN in PD in a pig model. Large animals such as pigs serve as great models to study pathological phenotypes for human neurological diseases due to their physiological similarity to humans (Prather et al., 2013). Wang et al. successfully implemented the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system into the Bama miniature pig genome to concurrently target three distinct loci by co-injecting cas9 mRNA and single-guide RNAs (sgRNA) which target PARKIN, DJ-1, and PINK1 genes into pronuclear embryos (Wang et al., 2016). Immunofluorescence, western blotting and reverse transcription-polymerase chain reaction (RT-PCR) confirmed a significant reduction in expression of these genes compared to wild-type with a low incidence of off-target mutations via whole-genome sequencing. Despite the drawbacks to using large animals as a means of genetic modification, including the fact that it is a time-consuming and expensive procedure due to lack of embryonic stem cell (ESC) lines, the effective and specific biallelic knock-outs of genes makes this a valuable tool to study neurological disorders where other animal models have failed.

Along with studying large model organisms, the PD field has also taken advantage of using patient-derived induced pluripotent stem cells (iPSCs). IPSCs are a special type of stem cell in which human somatic cells are engineered and genetically altered to be differentiated into other types of cells in the body such as neuronal, cardiac or hepatic via distinct transcription factors (Bellin et al., 2012). IPSCs make an extraordinary model for studying human diseases because they can reveal phenotypic defects and are a renewable source. Chung et al. differentiated PARKIN/PINK1 mutant and normal iPSC and ESC lines of midbrain DA neurons such that all cell lines demonstrated properties consistent with development of midbrain DA neurons (Chung et al., 2016). Although PARKIN and PINK1-derived iPSCs showed abnormal mitochondria (enlarged and enhanced oxidative stress), they were not prone to cell death. When given an oxidative stress inducer, carbonyl cyanide m-chlorophenyl hydrazine (CCCP), PD iPSC cell lines were more susceptible to cell death and displayed atypical neurotransmitter homeostasis (Chung et al., 2016).

In summary, upon mitochondrial depolarization, PINK1 recruits PARKIN to the mitochondrial membrane and its phosphorylation permits proteasome-dependent degradation of damaged mitochondria and enhances cell survival by suppressing apoptosis. Using whole exome genome sequencing to identify PD patients with specific PARKIN mutations that demonstrate dysfunctional mitophagy, therapeutic interventions could be targeted to prevent oxidative stress or promote regeneration of these cells via stem cell technologies specifically to DA neurons of the midbrain.

Angelman syndrome (AS) is a neurodevelopmental disorder that is one of the most well studied RNDs. Although specific symptoms vary in individual cases, AS is characterized by intellectual disability, developmental delay, distinct behavioral patterns such as a happy demeanor with prolonged, inappropriate laughter and smiling, speech impairment, seizures, abnormal sleep patterns, and ataxia (Williams et al., 2006; Tan and Bird, 2016). AS occurs in about one out of every 12,000 births (Steffenburg et al., 1996). Over 90% of AS cases are caused by mutations in the E3 ligase, UBE3A or deletions in the 15q11-13 maternal region containing UBE3A (Figure 3 and Supplementary Table 2; Kishino et al., 1997; Matsuura et al., 1997; Sutcliffe et al., 1997). Similar to UBE3A, mutations in another E3 ligase, HERC2, also lead to AS-like phenotypes (Puffenberger et al., 2012).

UBE3A encodes a HECT E3 ligase that is imprinted specifically in neurons of the central nervous system. Imprinting is established and maintained through expression of a long noncoding RNA on the paternal allele called the UBE3A antisense (UBE3A-ATS) transcript. As a result, only maternal UBE3A is expressed in neurons (Albrecht et al., 1997; Rougeulle et al., 1998; Runte et al., 2001, 2004; Yamasaki et al., 2003; Landers et al., 2004). Epigenetic regulation of UBE3A is similarly conserved in rodents, which has allowed researchers to generate AS murine models to study this disorder (Jiang et al., 1998). Using genetic engineering techniques, mouse models for AS were created to generate a viable maternally inherited Ube3a null mutation and a conditional reinstatement model has been created to restore the Ube3a null mutation during development (Jiang et al., 1998, 2010; Miura et al., 2002; Silva-Santos et al., 2015). AS mice display many AS-relevant phenotypes which include motor deficits, seizure susceptibility, learning impairments, and altered sleep homeostasis (Jiang et al., 1998, 2010; Miura et al., 2002; Cheron et al., 2005; Colas et al., 2005; van Woerden et al., 2007; Mulherkar and Jana, 2010; Ehlen et al., 2015). AS mice also exhibit deficits in multiple forms of synaptic plasticity such as long-term potentiation (LTP), long-term depression (LTD), metabotropic glutamate receptor (mGluR)-dependent LTD, homeostatic scaling, and ocular dominance plasticity (Figure 5; Jiang et al., 1998; Weeber et al., 2003; Dindot et al., 2008; Yashiro et al., 2009; Sato and Stryker, 2010; Pastuzyn and Shepherd, 2017).

The anatomical changes in AS have also been studied in great detail. In vertebrates, changes in dendritic spine dynamics are associated with alterations in learning, plasticity, and behavior throughout development (Dindot et al., 2008; Silva-Santos et al., 2015; Valluy et al., 2015; Pastuzyn and Shepherd, 2017). Notably, the density of dendritic spines was found to be reduced in a postmortem human AS patient using a traditional method of Golgi staining (Jay et al., 1991). Similarly, Ube3a^m−/p+(AS) mice have a decrease in dendritic spines, an absence of normal induction of LTD and LTP in the visual cortex, and have defective ocular dominance plasticity (Dindot et al., 2008; Yashiro et al., 2009; Sato and Stryker, 2010). The use of two-photon microscopy has allowed researchers to increase the depth of imaging in living tissue and provide longitudinal changes in functional connectivity, cortical response, and neural activity with limited phototoxicity (Yang and Yuste, 2017). To observe changes in dendritic spine dynamics, AS mice were crossed with Thy1-GFP males (Feng et al., 2000; Kim et al., 2016). The resulting offspring of this cross allowed GFP expression in Layer 5 pyramidal neurons in both wild type (WT) and AS mice (Kim et al., 2016). Although dendritic spine density was not altered in AS mice, dendritic spine elimination was significantly increased during the end of the first month of postnatal life. However, enhanced spine elimination could be rescued when AS mice were deprived of visual experience by dark rearing (Kim et al., 2016).

Gross anatomical differences have been observed in multiple brain regions in the AS mouse model (Judson et al., 2017). AS mice exhibit microcephaly and have significant reductions in white matter tracts. These microstructural abnormalities were investigated using diffusion tensor imaging (DTI), a tool that provides unique information of the preferred orientation, myelination, and density in white matter specifically in axon bundles in vivo (Basser and Pierpaoli, 1996; Goodlett et al., 2009). Using electron microscopy, a decrease in axon caliber (reduced cross-sectional diameter) in myelinated axons was identified within the corpus callosum and sciatic nerves, which later revealed slower action potential rise kinetics compared to controls (Judson et al., 2017). Generally, microcephaly is linked to early neurological phenotypes such as hypotonia and seizures in infants (Fryburg et al., 1991). Therefore, the relationship between the deficits in postnatal brain growth and pathophysiology can be understood by examining the mechanism of this phenotype and the developmental consequences due to loss of UBE3A.

Optogenetics is another tool used to measure how neural populations affect the circuitry and function of the brain leading to behavioral phenotypes. This technique typically uses light to manipulate the activity of light-sensitive ion channels to spatially and temporally control cells in select brain regions (Klapoetke et al., 2014; Deisseroth, 2015; Yang and Yuste, 2017). Previously, a loss of UBE3A was found to enhance dopamine release in the mesoaccumbal pathway (Riday et al., 2012; Berrios et al., 2016). To evaluate the role of dopamine release in consummatory behavior, optogenetics was used to evaluate motivational behavior in a conditional AS model (Ube3a^FLOX/p+) by crossing these mice to those that express CRE recombinase specifically in tyrosine hydroxylase neurons (TH^CRE). These mice were then transduced with a CRE-dependent adeno-associated virus (AAV5)-channelrhodopsin-2 (H134R) fused to an enhanced yellow fluorescent protein (ChR2-eYFP) into the ventral tegmental area and an optical fiber was placed above the nucleus accumbens (Berrios et al., 2016). Mice were then trained on a specific schedule to nose-poke during optical stimulation. Mice with a loss of UBE3A in tyrosine-hydroxylase neurons demonstrated increased reward-seeking behavior via optical self-stimulation by suppressing the co-release of gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter, in a non-canonical pathway (Berrios et al., 2016).

The generation of the UBE3A reinstatement model has allowed researchers to define neurodevelopmental windows that may rescue AS-related phenotypes. A conditional reinstatement mouse model of Ube3a was created using a CRE-dependent reinstatement of maternal Ube3a (Ube3a^STOP/p+). Mice lacking maternal Ube3a displayed consistent impaired behavioral performance using a battery of behavioral tests (rotarod, marble burying, open field, nest building, forced swim test, and epilepsy) similar to the traditional AS mouse model (Silva-Santos et al., 2015). CRE-dependent reinstatement of UBE3A rescued motor deficits in adolescent mice. However, other AS behaviors such as anxiety, repetitive behavior, and epilepsy could only be rescued during early development (Silva-Santos et al., 2015).

Electrophysiology, specifically local field potential recordings, allow researchers to understand dynamic neural networks by measuring action potentials and graded potentials that reflect synaptic activity in the neural network (Herreras, 2016). Using this method, full recovery of hippocampal LTP was found at every time point of UBE3A reinstatement (Silva-Santos et al., 2015).

Using in vivo patch-clamp electrophysiology in the same mouse model above, AS mice were found to have increased excitability and reduced orientation tuning in regular-spiking GABA-ergic pyramidal neurons. However crossing Ube3a^STOP/p+ with Gad2-CRE mice to specifically reinstate UBE3A in interneurons could rescue orientation tuning (Wallace et al., 2017).

A UBE3A reporter mouse was initially developed to assess regions in which UBE3A was imprinted (Dindot et al., 2008). The UBE3A-Yellow fluorescent protein knock-in mouse (Ube3a^m+/pYFP) was used to identify compounds to unsilence the paternal Ube3a allele. The rationale behind this work was that the majority of AS individuals have a maternally inherited disruption of UBE3A unlike the paternal copy which is normal, but is not expressed due to epigenetic modifications (Lalande and Calciano, 2007). Using the Ube3a^m+/pYFP knock-in mouse in a high-content drug screen, topoisomerase inhibitors were found to unsilence the paternal Ube3a allele (Huang et al., 2012). Notably, the topoisomerase I inhibitor, topotecan, upregulated neuronal UBE3A expression in an AS mouse and downregulated Ube3a-ATS and Snrpn (Small Nuclear Ribonucleoprotein Polypeptide N) paternal gene expression in the brain in vivo (Huang et al., 2012). Another corresponding study also used Ube3a^m+/pYFP knock-in mice to test the efficiency of antisense oligonucleotides (ASOs) in depleting the Ube3a-ATS as another strategy to unsilence the paternal allele of UBE3A (Meng et al., 2015). The targeting of the paternal dormant allele provides a potential treatment option for AS by focusing on epigenetic modifications.

The development of AS patient-derived iPSCs has allowed researchers to study AS in a more human relevant context. (Chamberlain et al., 2010; Russo et al., 2015). These lines have been useful in studying the exact genomic disruption afflicted in AS patients and to understand human epigenetic UBE3A regulation to assist in identifying novel therapeutic strategies (Stanurova et al., 2016; Takahashi et al., 2017). AS-derived iPSCs have an increase in resting membrane potentials, decreased spontaneous synaptic currents, and a loss of LTP induction (Fink et al., 2017). These phenotypes could be rescued by treating AS-derived iPSCs with topotecan to unsilence the UBE3A paternal allele. Treatment with topotecan resulted in an increase in UBE3A mRNA expression which led to a shift to a more hyperpolarized resting membrane potential and restoration of action potential firing to control levels promoting normal neuronal excitability (Fink et al., 2017). Importantly, targeting the silenced paternal allele in AS patients might alleviate AS-related phenotypes such as intellectual impairments and developmental delays by increasing synaptic events (Fink et al., 2017). In another study, targeting CGI methylation de novo via introducing a CpG-free cassette into AS patient-derived iPSCs was able to correct abnormal DNA methylation and result in normal UBE3A expression in DA neurons (Takahashi et al., 2017).

In parallel to the use of transgenic mice and human iPSCs, clinicians have sought to discover indicators specific to AS patients. One of the methods used in human cases was electroencephalography (EEG) for which AS patients tend to display theta rhythmicity, epileptiform spike-wave changes, and increased delta rhythmicity (Vendrame et al., 2012). Delta power in the AS mouse and in AS children was compared. Results from these studies demonstrated an increase in delta power during wakefulness and sleep in both AS mice and children with AS compared to matched controls (Sidorov et al., 2017). These studies reveal, that in AS, loss of UBE3A results in large-scale disruptions in rhythmic neural activity and shows that EEGs may serve as a potential biomarker for not just AS, but for other RNDs that have seizure phenotypes.

It is worth noting duplication of the 15q11-q13 region of the maternal chromosome harboring the UBE3A gene is also a common and highly penetrant factor of autism spectrum disorder (ASD) pathogenesis (Figure 1 and Supplementary Table 1), and an increased dosage of the UBE3A gene is associated with developmental delay and neuropsychiatric phenotypes (Cook et al., 1997; Glessner et al., 2009; Noor et al., 2015). UBE3A can function as both an E3 ligase and a transcriptional co-activator (Scheffner et al., 1993; Dindot et al., 2008). It is expressed monoallelically in neurons and is involved in many previously stated functions such as maintaining the proper level of dendritic branching, synapse formation, and controlling the frequency of mEPSCs (Lu et al., 2009; Greer et al., 2010; Margolis et al., 2010; Khatri et al., 2017). Because previous research has parsed out the importance of expression sensitivity of UBE3A in two disorders, developing drugs that can control the expression of either the paternal or maternal allele or those that modulate neuronal activity would be beneficial to this patient population. Moreover, due to the emergence of early symptoms in AS and ASD, individuals with UBE3A disruptions can be monitored throughout their lifetime to prevent or alleviate ongoing symptoms such as seizures or ataxia.

Mutation in E3 ligase genes are associated with a vast multitude of RNDs (Figure 3 and Supplementary Table 2). For the majority of RNDs, there is not strong supporting evidence to indicate a causal link between them. Many of the RNDs mentioned in detail below have implied a possible relationship between the RND and an E3 ligase through familial case studies by looking at probable critical regions on chromosomes to screen out less important genes (Sekine and Makino, 2017).

The 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.