Box 2

DOWN'S SYNDROME

Mental retardation can arise not only from loss or derangement of the function of a gene product but also from aberrant overproduction of a gene product. One example of mental retardation in this category is Down's Syndrome. Down's Syndrome results not from a genetic mutation but from aberrant chromosome duplication, specifically duplication of one copy of chromosome 21. For this reason, Down's Syndrome is also referred to as Trisomy 21. Because of this unique mechanism, Down's Syndrome is not a heritable disorder in the usual sense— it arises as an epigenetic phenomenon as part of the initial stages of chromosome replication during oocyte generation or oocyte fertilization.

Thus, Down's Syndrome arises as a result of the overproduction of proteins encoded by the genes on chromosome 21. Obviously the molecular basis of the syndrome is quite complex because of the plethora of gene products potentially involved. However, portions of chromosome 21 (region q22.2 specifically) that are critical for the development of Down's Syndrome have been identified. This region is referred to as the Down's Syndrome "critical locus" or "critical region." In part, this region was identified by characterizing Down's Syndrome patients who had not undergone complete duplication of chromosome 21. It is not clear at present precisely which genes in the critical region (or combination of genes) results in Down's Syndrome, and this is an area of active research at present.

Two genes on chromosome 21 are receiving particular attention at this point, because they are interesting in the context of known signal transduction mechanisms that we have been discussing as related to learning and memory. These are DYRK1 and superoxide dismutase (SOD, see references 35-37). DYRK1 is the human homologue to Drosophila minibrain kinase, which was identified in that species as a nervous system development-related gene. DYRK1 is homologous to members of the MAP kinase superfamily. The targets and mechanisms for regulation of DYRK1/minibrain kinase are unclear at present but are under active investigation. SOD is another interesting candidate. As we discussed in Chapters 6 and 7, superoxide has been implicated as a signaling molecule in hippocampal LTP, which is hypothesized to act through its capability to lead to autonomous PKC activation. Thus the interesting model arises that part of the defects in Down's Syndrome are due to overexpression of SOD and attenuation of superoxide signaling in the nervous system. In fact, transgenic animal models have indicated that this is a viable hypothesis, as SOD-overproducing mice have deficits in LTP and hippocampus-dependent memory formation.

A final chromosome 21 gene worth noting is the gene for amyloid precursor protein (APP). As we will discuss in more detail in Chapter 11, overproduction of the APP-derived product amyloid beta peptide likely leads to Alzheimer's Disease. Gene duplication of the APP gene in Down's Syndrome patients leads to overproduction of amyloid beta peptide, and unfortunately almost all Down's Syndrome patients who live past the age of 35 develop Alzheimer's Disease-like pathology as well.

App Molecule Alzheimer

FIGURE 5 The ubiquitination pathway. Covalent association of an E1 subunit with a single ubiquitin (Ub) molecule results in the ATP-dependent activation of Ub (Step 1). Ub is then transferred to an E2 subunit (Step 2). Association of the E2 and/or E3 plus the target protein results in the formation of a complex (Step 3). Complex formation results in the transfer of Ub by E2 either directly to the target protein or through the transfer of the Ub to an E3 (Step 4). E3 transfers successive UB molecules to the target protein forming poly-UB chains (Step 5). Reproduced from Weeber and Sweatt (46).

FIGURE 5 The ubiquitination pathway. Covalent association of an E1 subunit with a single ubiquitin (Ub) molecule results in the ATP-dependent activation of Ub (Step 1). Ub is then transferred to an E2 subunit (Step 2). Association of the E2 and/or E3 plus the target protein results in the formation of a complex (Step 3). Complex formation results in the transfer of Ub by E2 either directly to the target protein or through the transfer of the Ub to an E3 (Step 4). E3 transfers successive UB molecules to the target protein forming poly-UB chains (Step 5). Reproduced from Weeber and Sweatt (46).

the low-molecular-weight protein ubiquitin to substrate proteins (see Figure 5), a complex process involving other proteins that serve as intermediates and modulators of the final ubiquitination step. Ubiquitination, of course, by and large serves to control trafficking of proteins to the proteasome for degradation. However it is important to keep in mind that new functions for ubiquitination are being discovered. These new features play a signal transduction role conceptually similar to other post-translational processes like phosphorylation.

The Angelman Syndrome E6-AP E3 ligase is one member of a large family of around 60 different E3 ligases, each of which has different substrate selectivities. The E6-AP protein has a very restricted substrate specificity—known substrates include the p53 tumor suppressor protein,

E6-AP itself, and one additional protein of unknown function.

Expression of the UBE3A gene, in both human and mouse, exhibits a phenomenon called imprinting. Imprinting is a general term to describe epigenetic phenomena that can result in silencing the expression of a particular gene. In the case of the UBE3A gene, imprinting, through complex mechanisms that are not entirely clear at this point, results in selective silencing of the paternal copy of the gene in the hippocampus and cerebellum. The upshot of this is that the maternal copy of the gene is selectively expressed in these brain sub-regions. Thus, offspring who inherit a defective copy of UBE3A from their mother have a selective loss of the E6-AP ubiquitin ligase in their hippocampus and cerebellum. Put in genetics jargon, maternal deficiency (m-/p+) results in a subregion specific

Genetic Jargon Image

FIGURE 6 Selective deficit in context-dependent fear conditioning in ubiquitin ligase maternal deficiency mice. The E6AP ubiquitin ligase gene (the Angelman Syndrome gene) exhibits imprinting such that the maternal copy of the gene is selectively expressed in the hippocampus. Offspring of a female mouse deficient in the E6AP ubiquitin ligase thus have a selective hippocampal loss of the E6AP subtype of E3 ubiquitin ligase. In these experiments, wild-type mice or maternal deficiency mice were assessed for both contextual (Panel B) or cued (Panel C) fear conditioning 24 hours after training (Panel A). The same group of maternal deficiency mice (n = 12) and wild-type mice (n = 12) were assessed for both context- and tone-dependent freezing. (A) Wild-type and maternal deficiency mice showed comparable freezing during and after the tone and foot shock were administered. (B) Context dependent fear conditioning: Maternal deficiency mice displayed significantly less freezing than wild-type when returned to the test chamber 24 hours later. Significant p values (< .001 were seen at each sampling period and for the total data by X2 test. (C) Tone-dependent fear conditioning: Maternal deficiency mice and wild-type mice showed comparable freezing when presented with the tone in a novel context immediately after context-dependent testing. Tone is shown by horizontal bar and shock is indicated by arrows. Data and figures reproduced from Jiang et al. (40). Copyright Cell Press.

FIGURE 6 Selective deficit in context-dependent fear conditioning in ubiquitin ligase maternal deficiency mice. The E6AP ubiquitin ligase gene (the Angelman Syndrome gene) exhibits imprinting such that the maternal copy of the gene is selectively expressed in the hippocampus. Offspring of a female mouse deficient in the E6AP ubiquitin ligase thus have a selective hippocampal loss of the E6AP subtype of E3 ubiquitin ligase. In these experiments, wild-type mice or maternal deficiency mice were assessed for both contextual (Panel B) or cued (Panel C) fear conditioning 24 hours after training (Panel A). The same group of maternal deficiency mice (n = 12) and wild-type mice (n = 12) were assessed for both context- and tone-dependent freezing. (A) Wild-type and maternal deficiency mice showed comparable freezing during and after the tone and foot shock were administered. (B) Context dependent fear conditioning: Maternal deficiency mice displayed significantly less freezing than wild-type when returned to the test chamber 24 hours later. Significant p values (< .001 were seen at each sampling period and for the total data by X2 test. (C) Tone-dependent fear conditioning: Maternal deficiency mice and wild-type mice showed comparable freezing when presented with the tone in a novel context immediately after context-dependent testing. Tone is shown by horizontal bar and shock is indicated by arrows. Data and figures reproduced from Jiang et al. (40). Copyright Cell Press.

knockout of E6AP in both the mouse and human. Angelman Syndrome patients have a rare defect—a brain subregion-selective loss of a specific protein. This imprinting pattern and subregion-selective defect is, of course, consistent with the deficits observed in these patients, which selectively involve learning and motor control. It seems clear on its face that understanding the underlying pathology of Angelman Syndrome patients will give insights into the effects of hippocampal lesions in the human, much like studies of other patients such as H. M. have done. In the case of Angelman Syndrome patients, however, the lesion is genetic and not anatomical—in fact, there appear to be no discernable anatomical malformations in the CNS of AS patients. In Angelman Syndrome patients, the defect is, of course, also present from birth, in contrast to lesions such as those experienced by H. M.

After Kishino et al. discovered that the UBE3A gene was the Angelman Syndrome gene, Yong-hui Jiang in Art Beaudet's lab at Baylor College of Medicine developed ube3a knockout mice in order to generate a murine model for Angelman Syndrome. Yong-hui, who was a graduate student at the time, came down the hall to my lab to undertake a behavioral and electrophysio-logic characterization of his interesting mice. He studied the maternal deficiency mice, as opposed to other types of heterozygous or homozygous knockout animals, in order to model the human syndrome most accurately. Yong-hui discovered that mice with a maternal deficiency in ube3a ubiquitin ligase exhibited a selective deficit in contextual fear conditioning, consistent with the selective loss of hippocampal ubiquitin ligase due to the maternal imprinting (see reference 15 and Figure 6). The same mice did not exhibit any deficit in

FIGURE 7 Impairment of hippocampal LTP in ubiquitin ligase maternal deficiency mice. (A) Summary of field potential recordings from the CA1 region (mean ± SEM) from wild-type (n = 17 slices, 11 animals) and E6AP maternal deficiency mice (14 slices, 6 animals). Baseline measurements were taken for at least 20 minutes to confirm stability. Stimulation intensity was adjusted for responses that were ~50% of the maximal fEPSP. Two 100-Hz, 1-second stimuli were given at time 0. Each point is a 2-minute average of six individual fEPSP measurements. The inset presents extracellular field recordings from a representative experiment during a basline interval and 60 minutes after induction of LTP for a wild-type (m+/p+) and a maternal deficiency (m-/p+) slice. Scale bars for inset, 2 mV and 4 ms. (B & C) Baseline synaptic responses were similar for maternal deficiency and wild-type mice. Plots of fiber volley amplitude versus stimulus strength and fEPSP slope versus fiber volley amplitude revealed no difference between wild-type and maternal deficiency mice. Results shown are from nine slices for wild-type and eight slices for maternal deficiency mice. (D) Paired-pulse facilitation is not impaired in maternal deficiency mice. The magnitude of the second response is presented as a percentage of the first response. Results shown are from five slices for wild-type and seven slices for maternal deficiency animals. (E) Responses to tetanic stimulation are not different between maternal deficiency and wild-type mice. Total depolarization (the integral of the tetanic depolarization response) is presented for wild-type (n = 6, upper trace) and maternal deficiency (n = 6, lower trace) animals. Scale bars for inset, 2 mV and 90 ms. Reproduced from Jiang et al. (40).

cued conditioning. This serves as a nice positive control for the animals having normal capacity in the amygdala-dependent, hippocampus-independent component of the task.

In addition, hippocampal slices prepared from maternal deficiency mice exhibited a loss of long-term potentiation of Schaffer/collateral inputs in area CA1, but normal stimulus-response relationships and paired-pulse facilitation (see Figure 7). Thus, the hippocampus-dependent learning deficit apparently can be accounted for by a selective loss of LTP in the hippocampus— a striking finding considering that hip-pocampal baseline synaptic transmission, short-term plasticity, and hippocampal anatomy all appear normal in these mice.

In a broad sense these findings implicate the ubiquitin/proteosome pathway in mammalian associative learning and hip-pocampal long-term potentiation. This represents an interesting parallel to a variety of evidence demonstrating a role for the ubiquitin pathway in long-term facilitation and long-term memory in Aplysia. In this system, long-term behavioral sensitization is in part subserved by ubiquitin-mediated proteolysis of PKA regulatory subunits, which elicits long-term increases in PKA activity and long-term facilitation of neurotransmitter release (see Box 3). In the future, it will be interesting to determine to what extent the roles of the ubiquitin system in mammals parallels those in Aplysia.

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How To Win Your War Against Anxiety Disorders

How To Win Your War Against Anxiety Disorders

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