The Ash Twins Blog

A new book on Winston Churchill throws new light on the lionized figure:

As soon as he could, Churchill charged off to take his part in “a lot of jolly little wars against barbarous peoples.” In the Swat valley, now part of Pakistan, he experienced, fleetingly, an instant of doubt. He realized that the local population was fighting back because of “the presence of British troops in lands the local people considered their own,” just as Britain would if she were invaded. But Churchill soon suppressed this thought, deciding instead that they were merely deranged jihadists whose violence was explained by a “strong aboriginal propensity to kill.”

He gladly took part in raids that laid waste to whole valleys, writing: “We proceeded systematically, village by village, and we destroyed the houses, filled up the wells, blew down the towers, cut down the shady trees, burned the crops and broke the reservoirs in punitive devastation.” He then sped off to help reconquer the Sudan, where he bragged that he personally shot at least three “savages.”

The young Churchill charged through imperial atrocities, defending each in turn. When the first concentration camps were built in South Africa, he said they produced “the minimum of suffering” possible. At least 115,000 people were swept into them and 14,000 died, but he wrote only of his “irritation that kaffirs should be allowed to fire on white men.” …

As war secretary and then colonial secretary in the 1920s, he unleashed the notorious Black and Tans on Ireland’s Catholics, to burn homes and beat civilians. When the Kurds rebelled against British rule in Iraq, he said: “I am strongly in favor of using poisoned gas against uncivilized tribes.” It “would spread a lively terror.” (Strangely, Toye doesn’t quote this.)

Of course, it’s easy to dismiss any criticism of these actions as anachronistic. Didn’t everybody in Britain think that way then? One of the most striking findings of Toye’s research is that they really didn’t: even at the time, Churchill was seen as standing at the most brutal and brutish end of the British imperialist spectrum. This was clearest in his attitude to India. When Gandhi began his campaign of peaceful resistance, Churchill raged that he “ought to be lain bound hand and foot at the gates of Delhi and then trampled on by an enormous elephant with the new Viceroy seated on its back.” …

In 1943, to give just one example, a famine broke out in Bengal, caused, as the Nobel Prize-winning economist Amartya Sen has proven, by British mismanagement. To the horror of many of his colleagues, Churchill raged that it was their own fault for “breeding like rabbits” and refused to offer any aid for months while hundreds of thousands died.

From Baker and George, forthcoming:

We examine whether advertising increases household debt by studying the initial expansion of television in the 1950′s. Exploiting the idiosyncratic spread of television across markets, we use micro data from the Survey of Consumer Finances to test whether households with early access to television saw steeper debt increases than households with delayed access. Results indicate that exposure to television advertising increases the tendency to borrow for household goods and the tendency to carry debt. Television access is associated with higher debt levels for durable goods, but not with the total amount of non-mortgage debt.

The following is an extended excerpt from Floyd Bloom’s Feb 2010 review “The catecholamine neuron: Historical and future perspectives” from Progress in Neurobiology. It provides the best summary I’ve read on the physiology of the noradrenergic modulatory neurotransmitter system. Note that deficits in this neurotransmitter system are hypothesized to underlie attention deficit hyperactivity disorder (ADHD), and the mechanism of action of Ritalin (methylphenidate) and Adderol (amphetamine/dextroamphetamine) is to increase norepinephrine release from noradrenergic neurons. At a visiting-professor lunch, Dr. Aston-Jones told me that, non-intuitively, treatments for ADHD probably work by decreasing stimulus-induced norepinephrine release. A tonic increase of synaptic norepinephrine produced by the drug binds presynaptic alpha-2 autoreceptors and downregulates neurotransmitter release in response to unexpected stimuli. The consequence is a decreased likelihood of breaking one’s attention in response to unimportant environmental stimuli, and an improved ability to focus on the task at hand.

When do LC–NE neurons fire?

Although it was known that the locus ceruleus (LC) was the source of NE axonal projections across most of the neuraxis except the hypothalamus, direct electrophysiological observation of the firing patterns of these neurons was delayed until the mid-1970s, first in anesthetized rodents (Korf et al., 1974) and then in unanesthetized, freely behaving rats ([Aston-Jones and Bloom, 1981a] and [Aston-Jones and Bloom, 1981b]) and monkeys (Foote et al., 1980). The observations in the freely behaving animals greatly refined the anticipated functional repertoire of the LC neurons. In the anesthetized animal recordings, an early common feature was responsiveness to nociceptive stimuli, such as pressure on a paw or the tail; in fact, this nociceptive response was employed as one of the identifying electrophysiological parameters of LC neurons. From this response pattern, investigators predicted that LC discharge, and release of norepinephrine would generate anxiety as a functional consequence. However, when the discharge patterns of LC neurons were repeated in freely behaving rats and monkeys, these response patterns could be re-interpreted: instead of responding only to painful stimuli, LC neurons exhibited a more general and subtle pattern of activity: a slow, tonic, basal discharge rate, but with brief phasic responses to novel sensory stimuli of all kinds—visual, auditory, somato-sensory and gustatory. Furthermore, these neurons showed an interesting correlation between neuronal firing rate and wakefulness, with progressive diminution of already slow basal activity as the animals engagement with its environment decreased, and complete silencing of activity as the animal entered rapid eye movement sleep.

Just as the functional significances of the firing patterns of the DA neurons was refined from a simple ‘firing with reward’ concept to a more refined and biologically more profound functional consequence of error prediction, so has the insight into the behavioral consequences of the LC firing patterns been refined: from an initial, nociceptive-anxiety prediction, to more a general sensory events-alerting, with an attentional prediction.

(Aston-Jones and Cohen, 2005a) and (Aston-Jones and Cohen, 2005b) have taken these observations to a further refined interpretation with an integrative theory of locus coeruleus-norepinephrine function, that they term ‘adaptive gain and optimal performance’ invoking a more complex and specific role in the control of behavior than was previously thought. In their view, phasic LC activation is driven by the outcome of task-related decision processes and is proposed to facilitate ensuing behaviors and to help optimize task performance (exploitation). When the subject’s engagement in the task wanes, LC neurons revert to a slow tonic firing. They further note that in the non-human primate (and presumably also in man) the LC receives substantive inputs from the anterior cingulate cortex and the orbitofrontal cortices. As mentioned earlier, in the now-classic immunohistochemical mapping studies of Lewis and Morrison (see [Lewis et al., 1986] and [Lewis and Morrison, 1989]), these are precisely the forebrain regions with the most dense innervation by LC fibers, and are forebrain regions later shown by (Aston-Jones and Cohen, 2005a) and (Aston-Jones and Cohen, 2005b) and their co-workers that also send direct afferent connections to the LC neurons.

Aston-Jones and Cohen further propose that it is the output of these cortical afferent systems, known to monitor task-related utility that confers on the LC’s broad output the capacity to optimize utility on both short and long timescales. Cohen and his co-workers have expanded our views of these LC discharge patterns together with the post-excitatory refractory period attributed to noradrenergic collateral signals within the locus coeruleus to attribute to these neurons a role in attentional blink, a temporary deficit in processing of a target stimulus following successful processing of a previous target (Nieuwenhuis et al., 2005). Those observations, together with analyses of human visual and auditory discrimination tasks have created a very plausible case for attributing the p300 event related potential recorded with scalp electrodes in man to the novelty detection and attention focusing consequences of LC activation. Furthermore, when one considers the relatively short latency with which the LC neurons react to a salient and unexpected sensory stimulus (around 100 ms), the firing of LC axons to the cortex would allow for the synchronous activity at the tempero-parietal junction that is presently thought to be the origins of the P300 (see Aston-Jones and Cohen, 2005a G. Aston-Jones and J.D. Cohen, Adaptive gain and the role of the locus coeruleus-norepinephrine system in optimal performance, J. Comp. Neurol. 493 (2005), pp. 99–110. View Record in Scopus | Cited By in Scopus (53)[Aston-Jones and Cohen, 2005a], [Aston-Jones and Cohen, 2005b] and [Corbetta et al., 2008]).

What does NE do to target neurons?

As has been noted in many prior reviews, the initially reported effects of iontophoretically administered norepinephrine was to suppress the spontaneous activity of cerebellar Purkinje cells, dentate granule and hippocampal pyramidal neurons, and cerebro-cortical pyramidal neurons ([Aston-Jones et al., 1998], [Berridge and Waterhouse, 2003] and [Foote et al., 1983]). More in depth analysis, however, revealed that while basal activity was suppressed, the responses to spontaneous or evoked afferents was enhanced, both for excitatory and for inhibitory inputs; these sorts of modulatory effects help establish in my mind, if not for others, the dynamic synaptic vocabulary for monoamine neurons that I have long envisioned (Bloom, 1973). While we have long attributed most of this ‘enabling’ action to the transductive consequences of the beta-receptor and intracellular generation of cyclic adenosine monophosphate (Bloom et al., 1975), there are also reports that the actions of locally applied NE in cerebellum and hippocampal formation can be better antagonized by systemic alpha blockers than by beta blockers (Staunton et al., 1988). This is clearly an inconsistency that deserves further attention. One possibility for this discrepancy is that alpha-1 adrenergic receptors, like D1 dopaminergic receptors have a higher affinity for the natural ligand and their antagonists than the beta-adrenergic receptor and the D2 receptors (see [Arnsten and Goldman-Rakic, 1987] and Arnsten et al., 1999 A.F. Arnsten, R. Mathew, R. Ubriani, J.R. Taylor and B.M. Li, Alpha-1 noradrenergic receptor stimulation impairs prefrontal cortical cognitive function, Biol. Psychiatry 45 (1999), pp. 26–31. Article | PDF (98 K) | View Record in Scopus | Cited By in Scopus (98)[Arnsten et al., 1999]).

See previous posts on history, symptoms, neurophysiology, genetics, molecular biology, targets of UbE3A , and systems pathophysiology of Angelman Syndrome.

The laughing, happy demeanor with little or no verbalization is the most distinctive, unusual, and interesting aspect of Angelman syndrome. At the current state of the field, neuroscience has no good way to study the neural basis of this fascinating phenotype. Outside the AS field, some attempts have been made to study the happiness circuit. In one functional MRI study, for example, professional actors placed themselves into a happy or sad affect while in the scanner (Pelletier et al 2003). Nonoverlapping loci within the same regions (orbitofrontal, left medial prefrontal, left ventrolateral prefrontal, left uncus, and right pons) were identified for each emotion.

Addiction researchers have identified hedonic circuits in the midbrain and striatum and circuits for negative affect in the extended amygdala and insula (Koob & Volkow 2010). In humans, primates, and rodents, similar behaviors are predictably associated with hedonic experience (Berridge & Kringelbach 2008). Although many brain regions demonstrate activity associated with reward, stimulation of only a few small hotspots by themselves increase the probability of these hedonic behaviors.  From stimulation experiments, it appears that only the nucleus accumbens shell, the ventral pallidum, and the pontine parabrachial nucleus are causative areas for pleasure (Berridge & Kringelbach 2008). Humans report that stimulation of the nucleus accumbens causes pleasurable sensations, and rodents will voluntarily stimulate the nucleus accumbens until they die. Infusion of opioids or cannabinoids into these hotspots prior to stimulation multiplies the hedonic reaction. The ventral pallidum may be the most important node in the pleasure circuit, as it is only by lesion to this region that hedonic reactions to pleasurable stimuli can be abolished.  An interesting hypothesis is that in AS, circuit malfunction leads to hyperactivity in these areas. Measuring the fMRI BOLD activity in AS patients or recording electrical activity in the AS mouse model in these pleasure-linked areas would test this hypothesis.

Otherwise, we can philosophize: Do these patients behave this way because they are locked into a perpetual state of joy, or are their behavioral circuits so degraded that the only one that is intact is happy laughter? The subjective feeling of pleasure (mood) and the behavioral signs of pleasure (affect) are dissociable, and it is possible that in AS the latter occurs without the former. Circumstantial evidence that AS patients do experience authentic joy comes from the fact that their laughter and happy affect are not random, but tied to things that a child may find humorous; slap-stick humor, for example, is especially popular among AS patients (Clayton-Smith & Laan 2003). Given the mild neuropathological findings of AS and the rescue of neurological deficits in the AS mouse by a single-amino acid CaMKII mutation, one may be sanguine that drugs targeting these pathways or gene therapy restoring maternally-imprinted UbE3A could significantly ameliorate the symptoms of AS. In this scenario, we would be able to ask cured AS patients about their internal state and determine if they really had been in a permanent state of gaiety. And if that were the case, would a cure be ethical?

Berredge K, Kringelbach M (2008) “Affective neuroscience of pleasure: reward in humans and animals.” Psychopharmacology 199:457–480

Clayton-Smith J, Laan L. (2003) “Angelman syndrome: a review of the clinical and genetic aspects.” J Med Genet. 40(2):87-95. Review.

Koob GF, Volkow ND. (2010) “Neurocircuitry of addiction.” Neuropsychopharmacology. 35(1):217-38.

Pelletier M, Bouthillier A, Lévesque J, Carrier S, Breault C, Paquette V, Mensour B, Leroux JM, Beaudoin G, Bourgouin P, Beauregard M. (2003) “Separate neural circuits for primary emotions? Brain activity during self-induced sadness and happiness in professional actors.” Neuroreport. 14(8):1111-6.

See previous posts on history, symptoms, neurophysiology, genetics, molecular biology, and targets of UbE3A in Angelman Syndrome.

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 the proteins 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 the known functions of the ubiquitination targets of UBE3A. The three most relevant targets are 1) Arc – a protein which regulates synaptic strengths in response to neuronal activity;  2) Ephexin5 – a rho-GEF which regulates cytoskeletal remodeling to restrict synapse number;  and 3)Sacsin – A proteasome-related protein which is important in large neurons of the motor system.

Although the advances in our molecular and genetic understanding of Angelman Syndrome (AS) are commendable, what these changes mean for organ systems and the organism is not obvious. This disconnect is not unique to AS pathophysiology. It is problematic for investigators studying all neurobiological diseases to link identified molecular lesions with neural and behavioral dysfunction. To alleviate this situation, investigators study animal models of disease at the systems (in this case, the nervous system) level.

Fortunately for those interested in AS, a heterozygous ubiquitin E3A (UbE3A) knockout inherited from the mother recapitulates a neurodevelopmental disorder approximating AS in mice (Jiang et al 1998). These mice exhibit motor dysfunction, sound-induced seizures, an abnormal EEG (large amplitude 3 Hz spike wave activity), defective contextual fear-conditioning, and impaired hippocampal LTP. The brain appears normal with standard neuroanatomical and histological techniques. P53 levels are very high in the cells of these mice, alluding to the possibility that UbE3A can ubiquitinate p53 even in the absence of the viral E6 protein (see previous, molecular biology). Miura et al noted mostly the same pattern of findings in their independently-generated UbE3A maternal-KO mouse, but did not find increased p53 levels (2002).

Dindot et al engineered mice to express yellow fluorescent protein (YFP) fused to UbE3A in neurons (2008). In these mice, it was shown that UbE3A localizes to synapses and the nucleus, and that while neurons only use the maternal allele, glia use both the paternal and maternal allele of UbE3A (see genetics). Dendritic arbors were simplified in these mice, with abnormally small hypervariable spines and decreased spine density (Dindot et al 2008). Abnormal synchronous (epilepsy-like) oscillations in the cerebellum of these mice are rescued by gap junction blockers, suggesting that UbE3A may regulate the electrical coupling between cells (Cheron et al 2005). It was recently discovered that these mice also have decreased neurogenesis in the dentate gyrus of the hippocampus, which could have implications for the abnormal learning and LTP in these mice (Mardirossian et al 2009).

Yashiro et al analyzed the visual cortex of AS mice in greater detail (2009). In whole cell recordings from L2/3 pyramidal neurons, they noted a decreased frequency of miniature excitatory postsynaptic potentials (mEPSCs) compared to wild-type (WT) with no change in mEPSC amplitude. The authors also noted plasticity abnormalities in several paradigms. Dark rearing (raising animals in darkness), for example, decreased mEPSC frequency in WT but not in the KO. In addition, decreased long-term potentiation (LTP) and long term depression (LTD) were identified at the L4 to L2/3 synapse, a decrease which progressed in adulthood. Interestingly, LTP and LTD could be induced in dark-reared animals; it was not until normal sensory experience occurred that plasticity was impaired. Furthermore, plasticity could be restored by dark-rearing the mice in adulthood. Monocular deprivation experiments like that described in the previous section for Arc KO mice revealed an identical pattern – no ocular dominance reorganization in favor of the spared eye occurred in AS mice.

UbE3A-maternal-KO mice demonstrate increased levels of inhibitory phosphorylation on CaM kinase II in neurons (Weeber et al 2003). Prevention of this inhibitory phosphorylation by mutation of CaMKII’s threonines 305 and 306 rescues the mouse’s neurological deficits (van Woerden et al 2007). This surprising result is especially relevant given the recent finding that the activity-dependent translocation of proteasomes into dendritic spines depends on CaMKIIα acting as a scaffolding protein (Bingol et al 2010). This activity-dependent translocation is abolished by the T305/306 mutation. From these findings we can formulate this model: In AS, proteasomes translocate to the dendritic spine normally, but important degradation targets like Arc and Ephexin5 are not ubiquitinated and do not enter the proteasome. Some proteins are degraded while UbE3A’s targets remain, which by some mechanism leads to the inhibitory phosphorylation of CamKIIα. This phosphorylation encourages more proteasome to enter the spine, which leads to more aberrant degradation. This vicious cycle produces long, thin, abnormal spines (Dindot et al 2008) which cannot change in response to experience (Yashiro et al 2009). Prevention of CaMKIIα’s inhibitory phosphorylation breaks this cycle by suppressing the translocation of proteasomes into synapses. With fewer spine proteasomes, the UbE3A non-targets are protected from degradation relative to UbE3A targets, and a proteomically-balanced synapse is restored.

Bingol B, Wang CF, Arnott D, Cheng D, Peng J, Sheng M. (2010) “Autophosphorylated CaMKIIalpha acts as a scaffold to recruit proteasomes to dendritic spines.” Cell. 140(4):567-78.

Cheron G, Servais L, Wagstaff J, Dan B. (2005) “Fast cerebellar oscillation associated with ataxia in a mouse model of Angelman syndrome.” Neuroscience 130:631–637.

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.

Jiang, Y., Armstrong, D., Albrecht, U., Atkins, C. M., Noebels, J. L., Eichele, G., Sweatt, J. D., & Beaudet, A. L. (1998) “Mutation of the Angelman ubiquitin ligase in mice causes increased cytoplasmic p53 and deficits of contextual learning and long-term potentiation.” Neuron 21, 799–811

Mardirossian S, Rampon C, Salvert D, Fort P, Sarda N. (2009) “Impaired hippocampal plasticity and altered neurogenesis in adult Ube3a maternal deficient mouse model for Angelman syndrome.” Exp Neurol. 220(2):341-8.

Miura K, Kishino T, Li E, Webber H, Dikkes P, Holmes GL, Wagstaff J. (2002) “Neurobehavioral and electroencephalographic abnormalities in Ube3a maternal-deficient mice” Neurobiol Dis 9:149–159.

van Woerden, G.M., Harris, K.D., Hojjati, M.R., Gustin, R.M., Qiu, S., de Avila Freire, R., Jiang, Y.H., Elgersma, Y. and Weeber, E.J. (2007) “Rescue of neurological deficits in a mouse model for Angelman syndrome by reduction of alphaCaMKII inhibitory phosphorylation.” Nat. Neurosci., 10, 280–282.

Weeber, E.J., Jiang, Y.H., Elgersma, Y., Varga, A.W., Carrasquillo, Y., Brown, S.E., Christian, J.M., Mirnikjoo, B., Silva, A., Beaudet, A.L. et al. (2003) “Derangements of hippocampal calcium/calmodulin-dependent protein kinase II in a mouse model for Angelman mental retardation syndrome.” J. Neurosci., 23, 2634–2644.

Yashiro, K., et al., Ube3a is required for experience-dependent maturation of the neocortex. Nat Neurosci, 2009. 12(6): p. 777-783.

Eisenegger et al (Nature, 2010) find that

the sublingual administration of a single dose of testosterone in women causes a substantial increase in fair bargaining behaviour, thereby reducing bargaining conflicts and increasing the efficiency of social interactions. However, subjects who believed that they received testosterone—regardless of whether they actually received it or not—behaved much more unfairly than those who believed that they were treated with placebo. Thus, the folk hypothesis seems to generate a strong negative association between subjects’ beliefs and the fairness of their offers, even though testosterone administration actually causes a substantial increase in the frequency of fair bargaining offers in our experiment.

Yamagishi and Shinada (PNAS 2009) find that

certain players of an economic game reject unfair offers even when this behavior increases rather than decreases inequity. A substantial proportion (30–40%, compared with 60–70% in the standard ultimatum game) of those who responded rejected unfair offers even when rejection reduced only their own earnings to 0, while not affecting the earnings of the person who proposed the unfair split (in an impunity game). Furthermore, even when the responders were not able to communicate their anger to the proposers by rejecting unfair offers in a private impunity game, a similar rate of rejection was observed. The rejection of unfair offers that increases inequity cannot be explained by the social preference for inequity aversion or reciprocity; however, it does provide support for the model of emotion as a commitment device. In this view, emotions such as anger or moral disgust lead people to disregard the immediate consequences of their behavior, committing them to behave consistently to preserve integrity and maintain a reputation over time as someone who is reliably committed to this behavior.

Aharoni et al (2007) find that psychopathic personalities do not experience retributive feelings in response to stories about crimes:

Participants who rated either high or low in psychopathic traits read stories about a homicide. These stories were designed to evoke both retribution and the consequentialist motive of behavior control by varying, respectively, criminal intent and likelihood of recidivism. The participants then recommended a length of confinement for the offender. Individuals high in psychopathic traits were uniquely insensitive to retributive cues, and they were particularly consequentialist in their punishment of criminal offenders.

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.

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

Animal cells have evolved two systems to degrade excess, aged, and abnormal proteins: the ubiquitin-proteasome system and the lysosome (Tai & Schuman 2008). The ubiquitin-proteasome system is responsible for the degradation of most intracellular proteins, while the lysosome degrades most membrane and extracellular proteins.

The ubiquitin-proteasome system mediates essential functions in the nervous system (Tai & Schuman 2008). For example, proteasome inhibitors cause retrograde amnesia in rodents. Ubiquitination and proteasome pathways are essential for the candidate molecular mechanism for memory known as long term potentiation (LTP). Neurons, with their exceptionally large cell volumes and long life (most neurons in the brain are as old as the organism), necessitate especially efficient protein turnover and recycling.

Dysfunction of the ubiquitin-proteasome system has been implicated in the neurodegenerative disorders, such as Alzheimer disease, Parkinson disease, Huntington disease, amyothrophic lateral sclerosis (ALS, a.k.a. Lou Gehrig Disease), and the spinocerebellar ataxias (Lehman et al 2009). In these syndromes aggregates of misfolded proteins collect inside neurons and become resistant to proteasomal degradation. These misfolded proteins may actively inhibit the proteasome, or an upstream mechanism may disrupt proteasome function and permit aggregate formation. Less intuitively, the ubiquitin-proteasome pathway has been incriminated in the autism spectrum disorders (Glessner et al 2009; Lehman et al 2009). This situation makes more sense in light of the recent emergence of the hypothesis that autism is primarily a disorder of protein translation and degradation at dendritic spines (Geschwind 2008, Kelleher & Bear, 2008). Most relevant to the current work, the ubiquitin-proteasome pathway is perturbed in Angelman syndrome.

The proteasome is a 2.5 megadalton macromolecular machine that degrades intracellular proteins (Navon & Clechanover 2009). It consists of a 19S regulatory particle, which binds to ubiquitin and ubiquitin-like moieties to regulate which proteins are degraded, and the 20S core particle, which mediates the amino acid-by-amino acid disassembly of peptides. Proteasomes localize to dendritic spines to precisely regulate synaptic protein content after heightened synaptic activity (Tai & Schuman 2008), and it has recently been shown that this process requires CaM kinase IIα (CaMKIIα) functioning as a scaffolding protein (Bingol et al 2010). As genetic manipulation of CaMKII can rescue the neurological phenotype of the mouse model for AS (van Woerden et al 2007), this molecular connection between CaMKII and the proteasome could represent a essential locus for disease (More in a future post).

Ubiquitination refers to the covalent addition of a 76-amino acid peptide called ubiquitin (called this for historical reasons- was identified in the 1970′s as a protein of unknown function present in all cells) to the ε-amino group of lysine residues on target proteins (Pickart 2001). Key to ubiquitination is the fact that ubiquitin has many lysine residues of its own, so it too can be ubiquitinated, leading to long chains of ubiquitin on targeted proteins. Ubiquitination modulates many key cellular processes by selectively targeting master regulatory proteins for proteasomal degradation. Although degradation is the classic and most common fate for a ubiquitinated protein, ubiquitination can influence proteins in other ways, too, including modulation of ribosomal function, DNA repair, the inflammatory response, and transcription factor binding. Relevant to neuroscientists, ubiquitination can mark a receptor or ion channel for endocytosis (Hicke 1999). Whether ubiquitination signals for degradation or something else probably depends on the length of the polyubiquitin chain, the subcellular localization of the substrate, and the topology of the moiety formed by the ubiquitin-substrate complex (Pickart 2001). For example, polyubiquitin chains linked by K48-G76 bonds are usually destined for proteasomal degradation, whereas chains linked by K63-G76 bonds are implicated in intracellular signaling. Deubiquitinating enzymes (DUBs) depolymerize ubiquitin chains, and can thus reverse a protein’s degradation-fate if there are no proteasomes in close proximity and/or the ubiquitin chain is not long enough (Tai & Schuman 2009).

A dozen ubiquitin-related covalent protein modifiers have been identified in addition to ubiquitin (Jentsch & Pyrowolakis 2000). Most have high homology to ubiquitin but instead of mediating degradation are involved in a wealth of other cellular processes. Sumoylation with SUMO-1 (small ubiquitin-related modifier), for example, can direct proteins to translocate into the nucleus at specific points during the cell cycle. Rubylation with RUB (related to ubiquitin) stabilizes scaffolding proteins for the formation of ubiquitination complexes.

Ubiquitination occurs in a three-step process catalyzed by three ligases: E1, E2, and E3 (Pickart 2001). First, E1 ligase forms a thiol ester with ubiquitin’s G76 carboxyl group, activating the ubiquitin C-terminus. Next, E2 ligase transfers the activated ubiquitin from E1 to E3. Last, E3 ligase catalyzes the covalent linkage of ubiquitin to a protein substrate. Polyubiquitin chain formation sometimes requires an additional E4 ligase. The organization of ligases is hierarchical: There is one E1; several E2s, each of which can load several E3s; and hundreds of E3s, each of which target a few substrate proteins.

E3 ligases therefore determine which proteins are ubiquitinated by recognizing highly specific structural motifs on their target proteins.  The ubiquitination signal recognized by a given E3 is frequently a short primary amino acid sequence. Unfortunately, high-throughput methods for identifying E3 substrates are unavailable, and the specific targets for most E3s are undefined (Tai & Schuman 2008).

E3 ligases subdivide into two classes based on their catalytic domain, the HECT ligases and RING ligases. The HECT (Homologous to E6-AP Carboxy Terminus) domain is a highly conserved E2-binding site. RING ( Really Interesting New Gene) refers to a double-zinc cross-brace structure that also specifically binds E2. The domains, although structurally quite distinct, do not appear to differ significantly in function (Pickart 2001).

By incredible coincidence, ubiquitin E3A ligase (UbE3A), the protein implicated in Angelman Syndrome, was the first HECT ligase to be studied in detail. It was discovered as the E3 ligase which is co-opted by human papillomavirus (HPV) to ubiquitinate and degrade the antineoplastic protein p53. The HPV protein which binds UbE3A to degrade p53 is called E6, and thus UbE3A is also called E6-associated protein (E6-AP).

Until very recently, the normal physiological protein substrates of UbE3A (i.e. when it is not in a complex with E6) were for the most part unknown. The Howley lab showed that HHR23A and the Src kinase Blk are ubiquitinated by UbE3A (Kumar et al 1999; Oda et al 1999), and Banks and Kuhne demonstrates that Mcm7 is ubiquitinated by UbE3A as well (1998).  In the March 2010 issue of Cell, Greer et al. identify the ubiquitination signal of UbE3A and report its protein substrates. In the key experiment, a hemagglutinin-tagged ubiquitin knock-in mouse was crossed with the UbE3A knockout and WT mice. Brain lysate was incubated with anti-hemagglutinin antibody to pull down ubiquitin-bound proteins, which were then identified by mass spectrometry. Any proteins that were pulled down in the WT prep but not the UbE3A prep were likely ubiquitinated specifically by UbE3A. Three novel protein substrates were identified: Arc, Ephexin5, and Sacsin.

UbE3A recognizes these proteins and attaches a ubiquitin to them, which causes them to be degraded. Loss of UbE3A, as in Angelman Syndrome, would prevent these proteins from being degraded and lead to an excess of them in cells. In the next post, I describe the function of these proteins in greater detail to help us think about what the effects of their faulty degradation would be.

Bingol B, Wang CF, Arnott D, Cheng D, Peng J, Sheng M. (2010) “Autophosphorylated CaMKIIalpha acts as a scaffold to recruit proteasomes to dendritic spines.” Cell. 140(4):567-78.

Geschwind DH (2008)“Autism: many genes, common pathways?” Cell. 135(3):391-5.

Glessner JT, Wang K, Cai G (2009) “Autism genome-wide copy number variation reveals ubiquitin and neuronal genes.” Nature 459:569–573

Greer PL, Hanayama R, Bloodgood BL, Mardinly AR, Lipton DM, Flavell SW, Kim TK, Griffith EC, Waldon Z, Maehr R, Ploegh HL, Chowdhury S, Worley PF, Steen J, Greenberg ME. (2010) “The Angelman Syndrome protein Ube3A regulates synapse development by ubiquitinating arc.” Cell. 5;140(5):704-16.

Hicke L. (1999) “Gettin’ down with ubiquitin: turning off cell-surface receptors, transporters and channels.” Trends Cell Biol.9(3):107-12. Review.

Jentsch S & Pyrowolakis G (2000) “Ubiquitin and its kin: how close are the family ties?” Trends Cell Bio 10.

Kelleher RJ, Bear MF. (2008) “The autistic neuron: troubled translation?”  Cell.  135(3):401-6.

Kuhne C, Banks L. (1998) “E3-ubiquitin ligase/E6-AP links multicopy maintenance protein 7 to the ubiquitination pathway by a novel motif, the L2G box.” J. Biol. Chem. 273:34203–9

Kumar S, Talis A, Howley PM. (1999) “Identification of HHR23A as a Substrate for E6-associated Protein-mediated

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Lehman NL (2009) “The ubiquitin proteasome system in neuropathology.” Acta Neuropathol. 118(3):329-47.

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Pickart CM (2001) “Mechanisms underlying ubiquitination.” Annu Rev Biochem. 70:503-33. Review.

Tai & Schuman (2008) “Ubiquitin, the proteasome and protein degradation in neuronal function and dysfunction” Nat Rev Neuro.

van Woerden, G.M., Harris, K.D., Hojjati, M.R., Gustin, R.M., Qiu, S., de Avila Freire, R., Jiang, Y.H., Elgersma, Y. and Weeber, E.J. (2007) “Rescue of neurological deficits in a mouse model for Angelman syndrome by reduction of alphaCaMKII inhibitory phosphorylation.” Nat. Neurosci., 10, 280–282.