The Ash Twins Blog

See previous posts on history, symptoms, neurophysiology, genetics, and molecular biology of Angelman Syndrome.

To briefly review, Angelman Syndrome (AS) is a neurological syndrome which causes patients to be excessively cheerful, giggling, and happy, and mentally retarded with very little or no language. AS occurs when the protein ubiquitin E3A ligase (UbE3A) is lost due to abnormal methylation/imprinting of a region of chromosome 15. UbE3A recognizes proteins and attaches a ubiquitin to them, which causes them to be degraded in the proteasome. Loss of UbE3A in Angelman Syndrome would prevent these proteins from being degraded and lead to an excess of them in cells. In the previous post, I described how Greer et al identified ubiquitination targets of UBE3A: Arc, Ephexin5, HHR23A, Sacsin, Blk, and Mcm7. In this post I consider the function of these proteins.

Arc

Arc/Arg3.1 (activity-regulated cytoskeleton-associated protein/ activity-regulated gene 3.1; henceforth referred to as Arc) has multiple roles in neuronal function (Bramham et al 2008; Bramham et al 2010).  It is an early-immediate gene, meaning that it is transcribed into messenger RNA (mRNA) in association with heightened neural activity (its promoter includes binding sites for cAMP response element-binding protein (CREB), serum response factor (SRF), and myocyte-enhancer factor-2 (MEF2 – a TF which also enhances UbE3A transcription)). Arc mRNA transcripts are shuttled to dendrites and are translated into protein at sites of increased synaptic activity. Arc then associates with endophilin and dynamin to induce the internalization of AMPA receptors by endocytosis, which will function to decrease the synapse’s strength – this process is hypothesized to underlie the candidate neurophysiological mechanism for forgetting known as long term depression (LTD). Arc is also believed to mediate the structural consolidation of long term potentiation (LTP). In the current model (Bramham et al 2008), high synaptic activity induces brain-derived neurotrophic factor (BDNF) release into the synaptic cleft. BDNF binds to the TrkB tyrosine kinase receptor on the postsynaptic membrane. Activated TrkB phosphorylates and activates cytoplasmic proteins which induce Arc transcription (making mRNA with the gene as a blueprint) and local translation (making protein with the mRNA as a blueprint), and Arc indirectly phosphorylates cofilin. Phosphorylated cofilin mediates the expansion of actin filaments (globular proteins which are chained together to form a major component of the cytoskeleton, the cell’s internal scaffold). The enlarged synaptic scaffolding stabilizes the receptors and machinery needed for a stronger synapse. Arc’s key role in LTD and LTP has been confirmed behaviorally and electrophysiologically (Plath et al 2006).

The activity of Arc is exquisitely regulated within neurons (Bramham et al 2008). For example, Arc translation is inhibited by fragile X mental retardation protein (FMRP – the protein which is dysfunctional in Fragile X syndrome [the most common single-gene cause of mental retardation]) at baseline. When synaptic concentrations of the excitatory neurotransmitter glutamate reach a high enough level, metabotropic glutamate receptors (mGluR1) are activated and block FMRP’s inhibition of Arc translation.  mGluR1 activation also induces Arc translation through a kinase: Eukaryotic elongation factor 2 kinase (eEF2K) dissociates from mGluR1 and phosphorylates eukaryotic elongation factor 2 (eEF2). EEF2 then selectively upregulates Arc translation while suppressing translation of other proteins (Park et al 2008). Arc mRNA is also regulated by nonsense-mediated degradation. The 3’ untranslated-region (UTR) of Arc is a target for RNA-binding proteins which speed its degradation. It has been suggested that together these mechanisms could allow for a situation in which each Arc mRNA transcript leads to the production of a single Arc protein.

Arc’s role in neural circuit function is less understood, but there are some interesting clues. McCurry and colleagues, for instance, have identified a key role for Arc in experience-dependent cortical plasticity (2010). In primary visual cortex (V1), Arc is primarily expressed in layer 2-4 pyramidal neurons; interestingly, layer 5 neurons lack Arc, as does the thalamus. In Arc knockout mice, topographic organization in V1 is indistinguishable from wild-type by intrinsic imaging, and visual-evoked responses are normal. Activity-dependent reorganization in V1, on the other hand, is nonexistent in Arc KO mice. Monocular deprivation – the experimental technique in which one eye is sutured shut for a few days during the period of high cortical plasticity (4-5 weeks-old in mice (Gordon 1997), 6 months- to 8 years-old in humans (Vaegan & Taylor, 1979)) – has no effect on V1 organization in KO mice; compare this to wild-type animals, in which this transient deprivation causes a dramatic enlargement of ocular dominance columns representing the open eye at the expense of cortical surface area representing the sutured eye. Similarly, repeated presentation of the same visual stimulus over several days increases the electrophysiological response to that stimulus in WT mice but not in Arc KO mice. The monocular deprivation plasticity is likely mediated by a mGluR-LTD mechanism, and the repeated-stimulus potentiation is likely mediated by LTP, so these experiments strongly implicate Arc in both of these processes. In another study, Wang et al demonstrated that in Arc KO mice, V1 neurons have broader tuning curves relative to WT (2006). Moreover, Arc KO mice have impaired learning and memory in the Morris water maze, contextual fear conditioning, and conditioned taste aversion (Plath et al 2006) Last, induction of LTP and LTD is impaired in the hippocampi of these mice (Plath et al 2006).

What do these experimental findings tell us about the pathophysiology of AS? AS patients probably have a dysregulated, hyperactive Arc which is not degraded after it performs its function (Greer et al 2010). Tonic Arc activity should lead to the functionally-contradictory decrease in AMPA receptor density and increase in synapse size and stability. The overall effect on synaptic strength could vary from synapse to synapse, but a gross dysregulation of individual synapse strengths would be inevitable. The implication for network function would be an inability to fine-tune and optimize synaptic connections. This model could be tested by generating an Arc-overexpressing transgenic mouse.

Ephexin5

Ephexin5 is an uncharacterized Ras homology (rho) guanine nucleotide exchange factor (rho-GEF). Rho-GEFs are a class of enzymes which activate rho GTPases by catalyzing the exchange of GDP for GTP (Attalieb et al 2010). Rho GTPases are a subclass of the Ras superfamily small GTPases, and their major role is regulation of actin cytoskeleton dynamics (Etienne-Manneville & Hall 2002). In addition to their activation by rho-GEFs, Rho GTPases are deactivated by GTPase activating proteins (GAPs), which catalyze the GTP-to-GDP transition, and inhibited by guanine dissociation inhibitors (GDIs), which lock GTPases in the GDP-bound inactive state. Ephexin 5’s role in intracellular cascades is not worked out, but the related rho-GEF ephexin1 has been shown to catalyze the GDP-to-GTP activation in the rho GTPases RhoA, Cdc42, and Rac1, is the downstream target of the EphA receptor, regulates growth cone dynamics in developing neurons, and is required for the morphological “plaque-to-pretzel” maturation of the neuromuscular junction (Shamah et al 2001; Shi et al 2010). Greer et al cite unpublished findings to claim that ephexin5 restricts the number of synapses formed by a neuron (2010).

Working from the unpublished premise that ephexin5 restricts synapse number, overactive Ephexin5 would be expected to decrease the number of synapses on neurons. This hypothesis comports with Dindot et al’s findings in mouse maternal UbE3A knockouts that spine density is significantly decreased (2008). Dindot et al also found highly variable spine morphology compared to WT, which would also be expected given dysregulation of rho GTPase effects on the actin cytoskeleton.

HHR23A

HHR23A (human homologue of RAD23 A) is a unique protein that participates in both DNA repair and the proteasome system. In a complex with other proteins, HHR23a mediates nucleotide-excision repair, an adaptive response to ultraviolet (UV) DNA damage. The first 80 amino acids of HHR23A form a ubiquitin-like domain (UbL), which has high structural homology to ubiquitin and can bind to many of the same active sites as ubiquitin – the 19S proteasomal subunit, for example. It also has a ubiquitin-binding domain, which binds to ubiquitinated protein moieties. The current model is that the protein’s UbL domain binds and inhibits the proteasome, which prevents degradation of NER proteins bound to HHR23a. The significance of this protein’s expression for AS is unresolved.

Sacsin

Sacsin is another protein demonstrated to be a UbE3A ubiquitination target by Greer et al (2010). It is a little-studied protein, the mutation of which has been shown to cause spastic ataxia (Takiyama 2007). This autosomal recessive spastic ataxia was originally identified in the Charlevoix-Saguenay region of Quebec, where the mutant allele is exceptionally common due to a small founder population. As expected by the prominent motor-related symptoms, Sacsin is expressed primarily in large neurons of the motor system – for example, Betz cells in primary motor cortex and Purkinje cells in the cerebellum (Parfitt et al 2009). Like HHR23A, Sacsin has a UbL domain which interacts with the proteasome. Experiments suggest that Sacsin cooperates with Heat shock protein 70 (Hsp70) to stabilize protein folding and mediate proteasomal degradation of ataxia-related genes. Knockdown of Sacsin in heterologous cells increases the toxicity of polyglutamine-expanded ataxin-1 (the molecular lesion underlying spinocerebellar ataxia type 1)

Loss of ubiquitination would be expected to increase Sacsin levels in AS. As Sacsin appears to protect against toxic aggregates, it is not obvious how heightened Sacsin levels could lead to neuronal dysfunction. However, given Sacsin’s expression in large motor and Purkinje neurons, dysregulation of this protein is a prime candidate for the prominent motor and cerebellar dysfunction in AS.

Blk

Blk (B lymphoid tyrosine kinase) is a Src family nonreceptor tyrosine kinase which dimerizes with receptor tyrosine kinases to activate intracellular signaling cascades (Oda et al 1999). Src family kinases are involved in many cell processes; most relevant to AS, they are highly enriched in nerve growth cones (Helmke et al 1995). The active form is selectively ubiquitinated and degraded by UbE3A (Oda et al 1999). Dysregulated tyrosine kinase activity at growth cones could lead to abnormal neural circuit development.

Mcm7

MCM7 is a member of the minichromosome maintenance (MCM) complex (Lei 2005). The MCM complex moves with the DNA replication fork and likely performs a helicase function, separating the double-stranded DNA so it may be copied. The significance of this gene’s overactivity for AS is indeterminate.

It is probable that other UbE3A targets have yet to be identified, but the targets reviewed above are likely to play very important roles in AS’s pathogenesis.

Bramham CR, Worley PF, Moore MJ, Guzowski JF (2008) “The immediate early gene arc/arg3.1: regulation, mechanisms, and function.” J Neurosci 28(46):11760–11767

Bramham CR, Alme MN, Bittins M, Kuipers SD, Nair RR, Pai B, Panja D, Schubert M, Soule J, Tiron A, Wibrand K. (2010) “The Arc of synaptic memory.” Exp Brain Res. 200(2):125-40.

Dindot SV, Antalffy BA, Bhattacharjee MB, Beaudet AL. (2008) “The Angelman syndrome ubiquitin ligase localizes to the synapse and nucleus, and maternal deficiency results in abnormal dendritic spine morphology.” Hum Mol Genet 17:111–118.

Gordon JA (1997) “Cellular mechanisms of visual cortical plasticity: A game of cat and mouse” Learning and Memory, 4 (3):245-261.

Helmke S, Pfenninger KH. “Growth cone enrichment and cytoskeletal association of non-receptor tyrosine kinases” Cell Motil Cytoskeleton. 1995;30(3):194-207.

Lei M (2005) The MCM complex: its role in DNA replication and implications for cancer therapy. Curr Cancer Drug Targets. 5(5):365-80.

McCurry CL, Shepherd JD, Tropea D, Wang KH, Bear MF, Sur M. (2010) “Loss of Arc renders the visual cortex impervious to the effects of sensory experience or deprivation.” Nat Neurosci. 13(4):450-7.

Oda H, Kumar S, Howley PM. (1999). “Regulation of the Src family tyrosine kinase Blk through E6AP-mediated ubiquitination” PNAS 96:9557–62

Parfitt DA, Michael GJ, Vermeulen EG, Prodromou NV, Webb TR, Gallo JM, Cheetham ME, Nicoll WS, Blatch GL, Chapple JP. (2009) The ataxia protein sacsin is a functional co-chaperone that protects against polyglutamine-expanded ataxin-1. Hum Mol Genet. 18(9):1556-65. Epub 2009 Feb 10.

Park S, Park JM, Kim S, Kim JA, Shepherd JD, Smith-Hicks CL, Chowdhury S, Kaufmann W, Kuhl D, Ryazanov AG, Huganir RL, Linden DJ, Worley PF. (2008) “Elongation factor 2 and fragile X mental retardation protein control the dynamic translation of Arc/Arg3.1 essential for mGluR-LTD.” Neuron. 59(1):70-83.

Plath N, Ohana O, Dammermann B, Errington ML, Schmitz D, Gross C, Mao X, Engelsberg A, Mahlke C, Welzl H, Kobalz U, Stawrakakis A, Fernandez E, Waltereit R, Bick-Sander A, Therstappen E, Cooke SF, Blanquet V, Wurst W, Salmen B, Bosl MR, Lipp HP, Grant SG, Bliss TV, Wolfer DP, Kuhl D (2006) “Arc/Arg3.1 is essential for the consolidation of synaptic plasticity and memories.” Neuron 52(3):437–444

Shamah SM, Lin MZ, Goldberg JL, Estrach S, Sahin M, Hu L, Bazalakova M, Neve RL, Corfas G, Debant A, Greenberg ME. (2001) “EphA receptors regulate growth cone dynamics through the novel guanine nucleotide exchange factor ephexin.” Cell. 105(2):233-44.

Shi L, Butt B, Ip F, Dai Y, Jiang L, Yung W, Greenberg ME, Fu A, Ip NY (2010) “Ephexin1 Is Required for Structural Maturation and Neurotransmission at the Neuromuscular Junction” Neuron 65(2):204-216

Takiyama Y. (2007) Sacsinopathies: sacsin-related ataxia. Cerebellum. :1-7.

Vaegan & Taylor (1979). “Critical period for deprivation amblyopia in children.” Transactions of the Ophthalmological Society (UK) 99: 432-439.