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Huntington's disease: a synaptopathy?
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Huntington’s disease: a synaptopathy? Jia-Yi Li1, Markus Plomann2 and Patrik Brundin1 1
Section for Neuronal Survival, Wallenberg Neuroscience Center, Lund University, BMC A10, 221 84 Lund, Sweden Center for Biochemistry and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Cologne, Germany 2
Huntington’s disease (HD) is caused by a polyglutamine expansion in the protein huntingtin. In its terminal stage, HD is characterized by widespread neuronal death in the neocortex and the striatum. Classically, this neuronal death has been thought to underlie most of the symptoms of the disease. Accumulating evidence suggests, however, that cellular dysfunction is important in the pathogenesis of HD. We propose that specific impairment of the exocytosis and endocytosis machinery contributes to the development of HD. We also suggest that abnormal synaptic transmission underlies the early symptoms of HD and can contribute to the triggering of cell death in later stages of the disease. Huntington’s disease is clinically characterized by insidious onset and a slowly progressive course. It is hereditary and caused by a multiplication of CAG triplets in the IT15 gene, which results in the expansion of a polyglutamine region in the gene product huntingtin [1]. If the number of CAG triplets exceeds a crucial threshold of 39, disease invariably presents. Individuals suffering from HD gradually develop psychiatric disturbances, a motor movement disorder and cognitive deterioration. Symptoms typically begin at the age of 35 – 50 years, with substantial variation that depends, in part, on the length of the polyglutamine stretch [2]. Most commonly the number of CAG repeats is about 40 – 50, leading to disease onset in mid-life. By contrast, individuals with very long polyglutamine stretches – in excess of 70 glutamine residues – develop a juvenile form of the disease. Psychiatric symptoms, particularly depression, are frequent and often precede the onset of motor disturbance [3]. Individuals with HD typically show marked specific neuronal loss in the neocortex and in a defined region of the basal ganglia, namely, the neostriatum (caudate nucleus and putamen). In this region the most sensitive cells are the medium-sized spiny neurons. However, psychiatric and motor symptoms often precede detectable neuronal loss in HD, and many neurological syndromes proceed without obvious cell death [4]. It is thought that this might be due to a failure of neuronal functions such as cell – cell communication – a scheme that has been also suggested to apply to Alzheimer’s disease [5]. Here we discuss evidence suggesting that synaptic defects are a key factor in the pathogenesis of HD. Corresponding author: Jia-Yi Li ( [email protected]).
Biochemical properties and functions of huntingtin Huntingtin is highly conserved from Drosophila to mammals including humans, suggesting that it has a central role in cell functioning. Its broad subcellular distribution implies that it functions at several intracellular sites. Wild-type huntingtin is located mainly in the cytoplasm [6], is partly affiliated with membranous profiles [7] and binds to b-tubulin and microtubules [8,9]. Among the membrane compartments with which huntingtin is associated, synaptic vesicles [6,7], recycling endosomes, endoplasmic reticulum, Golgi complex and clathrin-coated vesicles [10] have an abundance of the protein. Taken together, the evidence suggests that wild-type huntingtin has a role in membrane trafficking in the cytoplasm and is also involved in microtubule-based axonal transport. In addition, huntingtin is found in the nucleus and seems to be vital for ontogenic development, because homozygous knockout mice show embryonic lethality [11]. Huntingtin-binding proteins Wild-type huntingtin is a very large protein of 350 kDa that might be involved in several functions through its numerous binding partners (Table 1). Among these partners are proteins with important roles in transcriptional regulation, intracellular trafficking and cytoskeletal organization. Many of the known huntingtin-binding proteins have roles in endocytosis, whereas comparatively few are involved in exocytosis. It is reasonable to propose that mutant huntingtin shows aberrant binding to individual interacting partners and, consequently, the function of the specific interacting protein is disrupted. In addition to affecting the normal interactions between huntingtin and its regular binding partners, the mutation can also lead to novel protein interactions. When huntingtin contains 37 or more consecutive glutamines, it misfolds and tends to form amyloid-like intracellular aggregates [12]. These aggregates can recruit several proteins, including those normally involved in synaptic function such as a-synuclein [13]. This might lead to a local depletion of components that are vital to the normal function of synapses. Furthermore, the presence of huntingtin aggregates might lead to saturation or structural inhibition of the ubiquitin– proteasome system – the chief cellular proteolytic pathway that normally processes misfolded proteins [14]. Functional inhibition of the ubiquitin– proteasome pathway might lead conceivably to an abnormal accumulation of both components of the
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Table 1. Huntingtin-interacting proteinsa Name
Binding region in huntingtin
Functions
Effects caused by huntingtin mutation
Refs
N-CoR
N terminus of htt171
Transcriptional regulation
[62]
Transcriptional co-repressor C-terminal binding protein WW domain proteins, HYPA, HYPB and HYPC CBP and p53
At PXDLS motif
Transcriptional regulation
CAG repeat length dependent binding Repressed transcription
N terminus, proline-rich region
Transcriptional regulation
N terminus
Transcriptional regulation
Sp1
N terminus
Transcriptional regulation
Gln-Ala repeat transcriptional activator (CA150) CIP4
Full-length huntingtin
Transcriptional regulation
N terminus
NF-kB/Rel/dorsal family transcription factor HAP1
C terminus
Involved in Cdc42 and WASp-dependent signal transduction Nuclear transport
HIP1
N terminus
HIP1-related/HIP12
Does not interact with huntingtin but can interact with HIP1
HIP14
N terminus
Hippi
Binds to HIP1
PACSIN I/syndapin,
N terminus, proline-rich region
Endophilins
Exon 1 protein, proline-rich region
PSD-95
N-terminal proline region
N terminus
Endosome – lysosome trafficking Clathrin-mediated endocytosis via binding to clathrin and AP2; promotes clathrin assembly; does not bind to actin Acts as functional link between clathrin and actin; promotes actin organization and clathrin assembly Intracellular trafficking and endocytosis Mediates apoptotic pathways.
Binds to dynamin, synaptojanin and N-WASp Bind to lipids; acts as a lysophosphatidic acid acyl transferase; interacts with dynamin and amphiphysins Binds and regulates the activity of glutamate receptors
[63]
Enhanced by lengthening of the adjacent glutamine tract Depleted from nucleus and present in aggregates; decreased transcription Enhanced interaction; disrupted interaction between Sp1 and TAFII130 Marked increase of CA150 expression Marked CIP4 overexpression and cell death induction of striatal neurons ?
[64]
[70]
Enhanced binding
[71]
Reduced interaction
[72 –74]
[46,65,66]
[51,67]
[68] [69]
[74,75]
Decreased interaction
[49]
Free HIP1 modulated by polyglutamine length in huntingtin; forms Hippi – HIP1 heterodimers and launches apoptosis Enhanced interaction
[76]
Enhanced binding to endophilin A3
[57,78]
Decreased interaction
[56]
[33,58,77]
a
Abbreviations: CA150, co-activator 150; CAG, Cys-Ala-Gly; CBP, CREB-binding protein; CIP4, cdc42-interacting protein 4; CREB, cAMP-response-element-binding protein; HAP1, Huntingtin-associated protein 1; HIP1, Huntingtin-interacting protein 1; HIP14, Huntingtin-interacting protein 14; Hippi, HIP1 protein interactor; htt, huntingtin; HYPA, htt yeast partner A; HYPB, htt yeast partner B; HYPC, htt yeast partner C; N-CoR, nuclear receptor co-repressor; PXDLS, Pro-Xaa-Asp-Leu-Ser; Sp1, specificity protein 1; TAF, TATA-binding protein-associated factor; WW, Trp-Trp.
endocytic and/or autophagic pathway and synaptic proteins that would be digested by this proteolytic pathway as part of the normal turnover of cellular protein [14,15]. Mutant huntingtin and synaptic dysfunction Distinct morphological abnormalities in neuronal dendrites are apparent as early degenerative changes both in transgenic mouse models of HD and in individuals affected with HD [16]. A marked decrease in the number of dendritic spines and a thickening of proximal dendrites has been observed before cell death is detectable [17]. Clinically, some individuals with genetically confirmed HD have been reported to develop severe motor and cognitive deficits and mental deterioration, even in the absence of widespread neuronal cell loss, as assessed by postmortem http://tmm.trends.com
examination of their brains [4]. Recent imaging studies have shown that there is progressive striatal and cortical dysfunction of the dopamine D2 receptor in individuals with HD [18]. Mutant huntingtin has been shown to impair directly the cellular machinery involved in synaptic transmission. Much insight has been gained by studies in the R6/1 and R6/2 transgenic mouse models of HD. These mice express exon 1 of the gene encoding human huntingtin containing a CAG repeat expansion of 115 or 150, respectively [19]. Corticostriatal glutamatergic fibers represent the principal excitatory input to the striatum [20]. Cellular pathology caused by mutant huntingtin in the presynaptic terminals might result in an increased release of glutamate. Alternatively, impaired clearance of glutamate
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from the synaptic cleft might increase glutamatergic neurotransmission. In both cases, striatal excitotoxicity could occur [21,22]. Intracerebral microdialysis has shown that depolarizing concentrations of potassium chloride increase the extracellular concentrations of glutamate substantially more in R6/1 mice than in wild-type mice [23] and, in particular, the glial glutamate transporter (GLT-1) is downregulated in R6/2 mice before any evidence of neurodegeneration [24]. This indicates that there is an impairment in glutamate transport and glutamate –glutamine cycling, and suggests that a defect in astrocytic glutamate uptake could contribute to the phenotype and to neuronal cell death in HD. Furthermore, mutant huntingtin binds to synaptic vesicles with higher affinity than does the wildtype form and inhibits the uptake of glutamate in synaptic vesicles in a dose-dependent manner [25]. With regard to the dopaminergic input to the striatum, intracerebral microdialysis experiments have detected substantially decreased concentrations of striatal extracellular dopamine in 16-week-old R6/1 mice [26]. Notably, this occurs at an age when there is no neuronal death in the substantia nigra or in the striatum. Moreover, postmortem tissue amounts of dopamine are unchanged in R6/1 mice at this age, indicating that their capacity to synthesize dopamine is still normal and suggesting that their synaptic function is perturbed [26]. Similarly, the striatal cholinergic system is also affected by mutant huntingtin in mouse models of HD. The evoked release of acetylcholine, its M2 autoreceptormediated maximum inhibition and its dopamine D2 heteroreceptor-mediated maximum inhibition are all substantially diminished in R6/2 mice as compared with wild-type mice [27]. In addition, the activity of choline acetyltransferase and the uptake of synaptosomal highaffinity choline decrease progressively with age [27]. R6/2 mice also show a significant reduction in long-term potentiation and long-term depression in the hippocampus [28]. In a different mouse model of HD, excitatory synapses in the hippocampus are impaired in their ability to sustain transmission during repetitive stimulation and there is indirect evidence that glutamate release is significantly reduced during higher frequency synaptic activation as compared with controls [29]. Taken together, there is much evidence for deficits that are located on the presynaptic side of the synapse in the machinery that regulates the release of neurotransmitters. How can mutant huntingtin impair normal neurotransmission? The molecular mechanisms underlying impaired neurotransmission in HD are still largely unknown. However, recent studies have provided important clues by showing that several synaptic vesicle proteins that are essential for synaptic transmission are abnormal in individuals with HD and HD transgenic mice [30 – 33]. Normal synaptic transmission is regulated by a large group of synaptic vesicle proteins that have key roles in membrane trafficking in exocytotic or endocytotic processes [34,35]. Alterations in the expression, post-translational modification and patterns of protein – protein interaction http://tmm.trends.com
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of these proteins have important implications for the function of neurons. Progressive loss of neuronal complexin II, a protein that regulates fusion processes between synaptic vesicles and the plasma membrane, has been shown in cells expressing mutant huntingtin, in R6/2 transgenic mice and in the brains of individuals with HD [30,31]. Furthermore, overexpression of complexin II in cultured cells expressing mutant huntingtin rescues the cells from premature death [36]. Additional evidence for changes in synaptic function in mouse models of HD comes from observations of an abnormal phosphorylation state of synapsin I in the striatum and the cerebral cortex of R6/2 transgenic mice. Such changes do not result from modifications at the level of protein expression [32], however, which suggests that an early impairment in synapsin phosphorylation/ dephosphorylation might alter synaptic vesicle trafficking and lead to defective neurotransmission in HD. Furthermore, PACSIN 1/syndapin, a neurospecific phosphoprotein with a central role in synaptic vesicle and receptor recycling, interacts with huntingtin. This interaction is dependent on the length of the polyglutamine repeat and is enhanced by the huntingtin mutation. In the presence of mutant huntingtin, PACSIN 1 seems to be removed from synapses and neuronal processes [33]. Several lines of mice with inactivated genes encoding synaptic vesicle proteins and/or huntingtin-interacting proteins show neuropathological changes. Among these, HIP1 knockout mice develop tremor and gait ataxia. The mice show a decreased assembly of endocytic protein complexes and profound defects in the internalization of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors containing glutamate receptor 1 (GluR1) [37]. In Drosophila, an absence of endophilin markedly impairs endocytosis in neuromuscular junctions and in synapses in the central nervous system [38,39]. Deletion of complexin II has been observed in different models of HD [30,31,36]. It has been shown that, although mice deficient for complexin II show no obvious neurological changes, long-term potentiation in both the CA1 and the CA3 regions of the hippocampus is impaired [40]. By contrast, homozygous mice deficient for complexin I develop severe gait ataxia, suffer from sporadic seizures and die 2– 4 months after birth, also in the absence of any obvious morphological brain changes [41]. Mice lacking both complexin I and complexin II die perinatally, even though there are no changes in brain cytoarchitecture [41]. These observations further strengthen the hypothesis that the dysfunction of synaptic vesicle proteins might be important in early events associated with HD. How does mutant huntingtin lead to an abnormality of synaptic vesicle proteins in HD? The mechanisms underlying changes in synaptic function caused by the presence of an expanded polyglutamine protein are still not well understood. As mentioned above, the expression of mutant huntingtin might lead to a general disruption of protein function, either by recruiting proteins to aggregates or by causing functional inhibition of the ubiquitin– proteasome system. Mutant huntingtin might also directly cause changes in protein expression.
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Microarray studies have shown that the patterns of changes in gene expression are complex in both cell and animal models of HD [42,43]. Although these patterns are not completely consistent among the different models, they show that mutant huntingtin can profoundly affect gene expression [44]. Huntingtin binds and interacts with several key transcription factors or coactivators (Table 1). There is evidence that the expanded polyglutamine stretch is crucial to its interaction with transcription factors such as TAFII130, a coactivator involved in transcriptional activation dependent on the cAMP-responsive element (CRE)-binding protein (CREB). The expanded polyglutamine stretch strongly suppresses CREB-dependent transcriptional activation [45], as well as depleting CREB-binding protein (CBP) from nuclear locations and specifically interfering with CBP-activated gene transcription [46]. Thus, mutant huntingtin can disrupt gene transcription through aberrant interactions with transcriptional factors. Moreover, some transcription factors need to be processed by the proteasome before they are activated [47]. Thus, there is more than one pathway by which mutant huntingtin could compromise the transcription of specific synaptic vesicle proteins, among which the expression of synaptoporin, N-ethylmaleimide sensitive factor (NSF) and synaptobrevin I is known to be markedly attenuated in HD (Figure 1) [42,43]. An alternative explanation for the disruption of synaptic function by mutant huntingtin is that the expanded polyglutamine stretch alters the interactions of wild-type huntingtin with other key proteins. This model can be viewed as a partial loss of normal function of the wild-type protein, leading to a disruption of the manner in which synaptic proteins function (Figures 1 and 2). After a (a) Normal Gene transcription
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protracted period (several years in humans) of failed synaptic protein function, cell death is eventually triggered. Botulinum toxins are known to disrupt synaptic function by cleaving the soluble NSF attachment protein (SNAP) receptor (SNARE) complex and, notably, this synaptic dysfunction can cause the subsequent degeneration of axons, dendrites and cell bodies of neurons, thereby leading to cell death [48]. Importantly, wild-type huntingtin interacts with several vesicle proteins that are vital for endocytosis. For mutant huntingtin, these interactions might be impaired and consequently might affect endocytic processes. For example, the binding affinity of huntingtin to PACSIN 1 is dependent on the length of the polyglutamine tract [33]; therefore, an increased interaction between the two proteins could lead to a sequestration of PACSIN 1 into non-synaptic aggregates. In addition, aberrant protein interactions could conceivably occur between pre-fibrillar species of huntingtin, which might be important in defective synaptic transmission. HIP14, another huntingtininteracting protein, has a role in intracellular transport processes and endocytosis [49]. A decreased interaction between mutant huntingtin and HIP14 in HD can impair endocytosis [49]. In analogy to synaptic vesicles, autophagic vacuoles also rely on the recycling of membranes through the formation of endosomes, and this process might be affected in HD. Thus, under conditions of increased oxidative stress, autophagic vacuoles accumulate excessively in the cytoplasm of R6/2 striatal neurons [15,50], indicating either an abnormality in the primary response to oxidative stress or a disturbance of vesicle cycling. Aggregates containing mutant huntingtin are usually located in the nucleus and cytoplasm, but they can also
(b) Huntington's disease Gene transcription –
Synaptic proteins
–
Synaptic proteins
Mutant huntingtin
+ or – Protein interaction
Protein interaction
Activation of cell death program
Transmitter release, synaptic vesicle cycling
Altered transmitter release and receptors
Excitotoxicity
Neurotransmission (including LTD/LTD)
Impaired neurotransmission
Neurodegeneration
Normal brain function
Motor symptoms, cognitive impairments, psychiatric disturbances TRENDS in Molecular Medicine
Figure 1. Principal cellular processes involved in the regulation of synaptic transmission. (a) Regulation under normal conditions. (b) Regulation under the pathological influences of mutant huntingtin. The most important take-home message is that not only does mutant huntingtin cause cell death directly, but also it can impair synaptic transmission at several levels, thereby leading to symptoms. http://tmm.trends.com
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Endocytosis
Exocytosis Actin
*
* Synapsin I
Synaptotagmin Synaptobrevin
HIP1R
N-WASp
HIP1
Rabphilin 3A
Clathrin-associated proteins
PACSIN 1 Rab3a
Complexin II
Endophilins Abp1p
Dynamin
SNAP-25
Syntaxin TRENDS in Molecular Medicine
Figure 2. Protein–protein interactions among key factors involved in exocytosis and endocytosis. Red arrows between individual proteins indicate specific binding interactions between those components. Dashed blue boxes indicate protein–protein interactions that potentially could be altered by mutant huntingtin, thereby disturbing endocytosis and exocytosis. Abbreviations: HIP1, Huntingtin-interacting protein 1; HIP1R, HIP1-related protein; N-WASp, neuronal Wiskott– Aldrich syndrome protein; PACSIN 1, protein kinase C and casein kinase in neurons 1.
appear in the axon and nerve terminals [25]. As discussed above, mutant huntingtin binds to synaptic vesicles with higher affinity than does wild-type huntingtin and inhibits the uptake of glutamate by synaptic vesicles [25], suggesting that it might be able to affect synaptic homeostasis directly. Presynaptic versus postsynaptic involvement In the above sections, we focused on the role of presynaptic mechanisms in the pathogenesis of HD symptoms. But are postsynaptic mechanisms also important in this process? Mutant huntingtin can downregulate several Sp1-dependent neuronal genes, including those encoding the dopamine D1, D2 and D3 receptors that are localized in both pre- and postsynaptic components [51]. In HD, mutant huntingtin selectively decreases the expression of N-methyl-D-aspartic acid (NMDA) receptors at presymptomatic stages [52]. At later stages, binding to individual ionotropic glutamate receptors is decreased and there is a less marked reduction in metabotropic glutamate receptor binding. In addition, there is a selective loss of kainic acid and AMPA binding in the frontal cortex [53]. In medium-sized spiny neurons of R6/2 HD transgenic mice, currents induced by the selective activation of NMDA receptors are enhanced, cytoplasmic Ca2þ concentration is increased and the frequency of spontaneous excitatory postsynaptic currents is raised [54,55]. Furthermore, wild-type huntingtin is associated with NMDA and kainate receptors through postsynaptic density 95 (PDS-95), whose Src homology domain 3 mediates the binding to huntingtin [56]. Thus, huntingtin could be involved in signal transduction immediately downstream of the neurotransmitter receptors. Notably, expanded polyglutamine stretches interfere with the ability of huntingtin to interact with PSD-95 and, in http://tmm.trends.com
turn, influence the normal function of NMDA and kainate receptors [56]. In addition, normal receptor recycling in nerve terminals is regulated by several proteins that also bind to huntingtin [33,57]. Proteins such as PACSIN 1 and endophilins are thought to participate in the regulation of receptor internalization by linking the actin polymerization machinery to endocytic protein complexes [58,59]. Mutant huntingtin might also impair proper receptor recycling in postsynaptic neurons. Finally, it is worth mentioning that selective reduction of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) has been observed in presymptomatic mouse models of HD [60]. DARPP-32 has a key role in many neurotransmitter pathways [61] and is involved in controlling receptors and ion channels, among others. DARPP-32 is involved not only in dopamine signal transduction, but also in signaling via many other neurotransmitters, including glutamate and serotonin. Therefore, a reduction of DARPP-32 in HD might contribute to the dysfunction of NMDA, AMPA and dopamine receptors and might disrupt normal synaptic transmission. Concluding remarks and future directions Little is known about the normal cellular function of huntingtin or how its function is altered by an expansion of polyglutamine. Recent studies suggest that the polyglutamine expansion might enable mutant huntingtin to corrupt normal gene transcription [51] and to disrupt normal intracellular trafficking and transmitter release [25,54]. But several issues remain with regard to how cellular dysfunction, including synaptic disturbances, can lead to neurodegeneration. Molecular genetic approaches will be useful for dissecting the roles of various factors in HD. Improved understanding of such interactions will pave the way for
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targeting one or more of the aberrant protein – protein interactions in novel therapies. Eventually, it might be feasible to develop drugs that alleviate the negative effect of polyglutamine expansions by interfering with specific pathways of gene expression or by altering undesirable protein – protein interactions. This could allow intervention in the disease process very much upstream of the events that eventually lead to cell death. The idea that we might be able to inhibit events in the pathogenetic cascade early on raises the exiting possibility that ultimately the development of clinical symptoms might be completely prevented. Note added in proof Very recently, it was demonstrated that nerve terminals in different HD animal models have far fewer synaptic vesicles [79]. Mutant huntingtin binds more tightly to synaptic vesicles compared with wild-type huntingtin; glutamate release is substantially reduced in the HD brain slice. These observations indicate impairment of synaptic function in HD.
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Acknowledgements ˚ sa Peterse´n and Ruben Smith for critically reading the We thank A manuscript. Work cited from our laboratories is supported by grants from the Swedish Research Council, the Hereditary Disease Foundation, the Swedish Society for Medicine, Crafoord Foundation and Hedlund Foundation, the Center for Molecular Medicine (CMMC; TP78) and Ko¨ln Fortune from the Medical Faculty of the University of Cologne. Some of the ideas presented in this article were stimulated by discussions in European Research Project (Concerted Action) entitled Early Pathogenetic Markers Of Slow Neurodegenerative Diseases (EPSND).
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vesicle budding at the Drosophila neuromuscular junction. EMBO J. 21, 1661 – 1672 Takahashi, S. et al. (1999) Reduced hippocampal LTP in mice lacking a presynaptic protein: complexin II. Eur. J. Neurosci. 11, 2359– 2366 Reim, K. et al. (2001) Complexins regulate a late step in Ca2þdependent neurotransmitter release. Cell 104, 71 – 81 Luthi-Carter, R. et al. (2002) Dysregulation of gene expression in the R6/2 model of polyglutamine disease: parallel changes in muscle and brain. Hum. Mol. Genet. 11, 1911 – 1926 Sipione, S. et al. (2002) Early transcriptional profiles in huntingtininducible striatal cells by microarray analyses. Hum. Mol. Genet. 11, 1953 – 1965 Cha, J.H. (2000) Transcriptional dysregulation in Huntington’s disease. Trends Neurosci. 23, 387 – 392 Shimohata, T. et al. (2000) Expanded polyglutamine stretches interact with TAFII130, interfering with CREB-dependent transcription. Nat. Genet. 26, 29 – 36 Nucifora, F.C. Jr et al. (2001) Interference by huntingtin and atrophin-1 with CBP-mediated transcription leading to cellular toxicity. Science 291, 2423 – 2428 Pahl, H.L. and Baeuerle, P.A. (1996) Control of gene expression by proteolysis. Curr. Opin. Cell Biol. 8, 340– 347 Williamson, L.C. and Neale, E.A. (1998) Syntaxin and 25-kDa synaptosomal-associated protein: differential effects of botulinum neurotoxins C1 and A on neuronal survival. J. Neurosci. Res. 52, 569 – 583 Singaraja, R.R. et al. (2002) HIP14, a novel ankyrin domain-containing protein, links huntingtin to intracellular trafficking and endocytosis. Hum. Mol. Genet. 11, 2815 – 2828 Petersen, A. et al. (2001) Expanded CAG repeats in exon 1 of the Huntington’s disease gene stimulate dopamine-mediated striatal neuron autophagy and degeneration. Hum. Mol. Genet. 10, 1243– 1254 Dunah, A.W. et al. (2002) Sp1 and TAFII130 transcriptional activity disrupted in early Huntington’s disease. Science 296, 2238 – 2243 Cha, J.H. et al. (1998) Altered brain neurotransmitter receptors in transgenic mice expressing a portion of an abnormal human Huntington’s disease gene. Proc. Natl. Acad. Sci. U. S. A. 95, 6480 – 6485 Wagster, M.V. et al. (1994) Selective loss of [3H]kainic acid and [3H]AMPA binding in layer VI of frontal cortex in Huntington’s disease. Exp. Neurol. 127, 70 – 75 Cepeda, C. et al. (2001) NMDA receptor function in mouse models of Huntington’s disease. J. Neurosci. Res. 66, 525– 539 Hansson, O. et al. (2001) Resistance to NMDA toxicity correlates with appearance of nuclear inclusions, behavioural deficits and changes in calcium homeostasis in mice transgenic for exon 1 of the Huntington gene. Eur. J. Neurosci. 14, 1492– 1504 Sun, Y. et al. (2001) Polyglutamine-expanded huntingtin promotes sensitization of N-methyl-D -aspartate receptors via post-synaptic density 95. J. Biol. Chem. 276, 24713 – 24718 Modregger, J. et al. (2003) Characterization of endophilin B1b, a brainspecific membrane-associated lysophosphatidic acid acyl transferase with properties distinct from endophilin A1. J. Biol. Chem. 278, 4160 – 4167 Kessels, M.M. and Qualmann, B. (2002) Syndapins integrate N-WASP in receptor-mediated endocytosis. EMBO J. 21, 6083– 6094 Tang, Y. et al. (1999) Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the b1-adrenergic receptor. Proc. Natl. Acad. Sci. U. S. A. 96, 12559 – 12564
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60 Bibb, J.A. et al. (2000) Severe deficiencies in dopamine signaling in presymptomatic Huntington’s disease mice. Proc. Natl. Acad. Sci. U. S. A. 97, 6809– 6814 61 Greengard, P. (2001) The neurobiology of slow synaptic transmission. Science 294, 1024– 1030 62 Boutell, J.M. et al. (1999) Aberrant interactions of transcriptional repressor proteins with the Huntington’s disease gene product, huntingtin. Hum. Mol. Genet. 8, 1647– 1655 63 Kegel, K.B. et al. (2002) Huntingtin is present in the nucleus, interacts with the transcriptional corepressor C-terminal binding protein, and represses transcription. J. Biol. Chem. 277, 7466 – 7476 64 Faber, P.W. et al. (1998) Huntingtin interacts with a family of WW domain proteins. Hum. Mol. Genet. 7, 1463– 1474 65 Steffan, J.S. et al. (2000) The Huntington’s disease protein interacts with p53 and CREB-binding protein and represses transcription. Proc. Natl. Acad. Sci. U. S. A. 97, 6763 – 6768 66 Wyttenbach, A. et al. (2001) Polyglutamine expansions cause decreased CRE-mediated transcription and early gene expression changes prior to cell death in an inducible cell model of Huntington’s disease. Hum. Mol. Genet. 10, 1829 – 1845 67 Li, S.H. et al. (2002) Interaction of Huntington’s disease protein with transcriptional activator sp1. Mol. Cell. Biol. 22, 1277– 1287 68 Holbert, S. et al. (2001) The Gln-Ala repeat transcriptional activator CA150 interacts with huntingtin: neuropathologic and genetic evidence for a role in Huntington’s disease pathogenesis. Proc. Natl. Acad. Sci. U. S. A. 98, 1811 – 1816 69 Holbert, S. et al. (2003) Cdc42-interacting protein 4 binds to huntingtin: neuropathologic and biological evidence for a role in Huntington’s disease. Proc. Natl. Acad. Sci. U. S. A. 100, 2712– 2717 70 Takano, H. and Gusella, J.F. (2002) The predominantly HEAT-like motif structure of huntingtin and its association and coincident nuclear entry with dorsal, an NF-kB/Rel/dorsal family transcription factor. BMC Neurosci. 3, 15 71 Li, X.J. et al. (1995) A huntingtin-associated protein enriched in brain with implications for pathology. Nature 378, 398 – 402 72 Wanker, E.E. et al. (1997) HIP-I: a huntingtin interacting protein isolated by the yeast two-hybrid system. Hum. Mol. Genet. 6, 487 – 495 73 Kalchman, M.A. et al. (1997) HIP1, a human homologue of S. cerevisiae Sla2p, interacts with membrane-associated huntingtin in the brain. Nat. Genet. 16, 44 – 53 74 Legendre-Guillemin, V. et al. (2002) HIP1 and HIP12 display differential binding to F-actin, AP2, and clathrin. Identification of a novel interaction with clathrin light chain. J. Biol. Chem. 277, 19897 – 19904 75 Engqvist-Goldstein, A.E. et al. (1999) An actin-binding protein of the Sla2/Huntingtin interacting protein 1 family is a novel component of clathrin-coated pits and vesicles. J. Cell Biol. 147, 1503– 1518 76 Gervais, F.G. et al. (2002) Recruitment and activation of caspase-8 by the Huntingtin-interacting protein Hip-1 and a novel partner Hippi. Nat. Cell Biol. 4, 95 – 105 77 Modregger, J. et al. (2000) All three PACSIN isoforms bind to endocytic proteins and inhibit endocytosis. J. Cell Sci. 113, 4511 – 4521 78 Sittler, A. et al. (1998) SH3GL3 associates with the Huntingtin exon 1 protein and promotes the formation of polygln-containing protein aggregates. Mol. Cell 2, 427 – 436 79 Li, H. et al. (2003) Abnormal association of mutant huntingtin with synaptic vesicles inhibits glutamate release. Hum. Mol. Genet. 12, 2021– 2030
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Huntington’s disease: a synaptopathy? Jia-Yi Li1, Markus Plomann2 and Patrik Brundin1 1
Section for Neuronal Survival, Wallenberg Neuroscience Center, Lund University, BMC A10, 221 84 Lund, Sweden Center for Biochemistry and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Cologne, Germany 2
Huntington’s disease (HD) is caused by a polyglutamine expansion in the protein huntingtin. In its terminal stage, HD is characterized by widespread neuronal death in the neocortex and the striatum. Classically, this neuronal death has been thought to underlie most of the symptoms of the disease. Accumulating evidence suggests, however, that cellular dysfunction is important in the pathogenesis of HD. We propose that specific impairment of the exocytosis and endocytosis machinery contributes to the development of HD. We also suggest that abnormal synaptic transmission underlies the early symptoms of HD and can contribute to the triggering of cell death in later stages of the disease. Huntington’s disease is clinically characterized by insidious onset and a slowly progressive course. It is hereditary and caused by a multiplication of CAG triplets in the IT15 gene, which results in the expansion of a polyglutamine region in the gene product huntingtin [1]. If the number of CAG triplets exceeds a crucial threshold of 39, disease invariably presents. Individuals suffering from HD gradually develop psychiatric disturbances, a motor movement disorder and cognitive deterioration. Symptoms typically begin at the age of 35 – 50 years, with substantial variation that depends, in part, on the length of the polyglutamine stretch [2]. Most commonly the number of CAG repeats is about 40 – 50, leading to disease onset in mid-life. By contrast, individuals with very long polyglutamine stretches – in excess of 70 glutamine residues – develop a juvenile form of the disease. Psychiatric symptoms, particularly depression, are frequent and often precede the onset of motor disturbance [3]. Individuals with HD typically show marked specific neuronal loss in the neocortex and in a defined region of the basal ganglia, namely, the neostriatum (caudate nucleus and putamen). In this region the most sensitive cells are the medium-sized spiny neurons. However, psychiatric and motor symptoms often precede detectable neuronal loss in HD, and many neurological syndromes proceed without obvious cell death [4]. It is thought that this might be due to a failure of neuronal functions such as cell – cell communication – a scheme that has been also suggested to apply to Alzheimer’s disease [5]. Here we discuss evidence suggesting that synaptic defects are a key factor in the pathogenesis of HD. Corresponding author: Jia-Yi Li ( [email protected]).
Biochemical properties and functions of huntingtin Huntingtin is highly conserved from Drosophila to mammals including humans, suggesting that it has a central role in cell functioning. Its broad subcellular distribution implies that it functions at several intracellular sites. Wild-type huntingtin is located mainly in the cytoplasm [6], is partly affiliated with membranous profiles [7] and binds to b-tubulin and microtubules [8,9]. Among the membrane compartments with which huntingtin is associated, synaptic vesicles [6,7], recycling endosomes, endoplasmic reticulum, Golgi complex and clathrin-coated vesicles [10] have an abundance of the protein. Taken together, the evidence suggests that wild-type huntingtin has a role in membrane trafficking in the cytoplasm and is also involved in microtubule-based axonal transport. In addition, huntingtin is found in the nucleus and seems to be vital for ontogenic development, because homozygous knockout mice show embryonic lethality [11]. Huntingtin-binding proteins Wild-type huntingtin is a very large protein of 350 kDa that might be involved in several functions through its numerous binding partners (Table 1). Among these partners are proteins with important roles in transcriptional regulation, intracellular trafficking and cytoskeletal organization. Many of the known huntingtin-binding proteins have roles in endocytosis, whereas comparatively few are involved in exocytosis. It is reasonable to propose that mutant huntingtin shows aberrant binding to individual interacting partners and, consequently, the function of the specific interacting protein is disrupted. In addition to affecting the normal interactions between huntingtin and its regular binding partners, the mutation can also lead to novel protein interactions. When huntingtin contains 37 or more consecutive glutamines, it misfolds and tends to form amyloid-like intracellular aggregates [12]. These aggregates can recruit several proteins, including those normally involved in synaptic function such as a-synuclein [13]. This might lead to a local depletion of components that are vital to the normal function of synapses. Furthermore, the presence of huntingtin aggregates might lead to saturation or structural inhibition of the ubiquitin– proteasome system – the chief cellular proteolytic pathway that normally processes misfolded proteins [14]. Functional inhibition of the ubiquitin– proteasome pathway might lead conceivably to an abnormal accumulation of both components of the
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Table 1. Huntingtin-interacting proteinsa Name
Binding region in huntingtin
Functions
Effects caused by huntingtin mutation
Refs
N-CoR
N terminus of htt171
Transcriptional regulation
[62]
Transcriptional co-repressor C-terminal binding protein WW domain proteins, HYPA, HYPB and HYPC CBP and p53
At PXDLS motif
Transcriptional regulation
CAG repeat length dependent binding Repressed transcription
N terminus, proline-rich region
Transcriptional regulation
N terminus
Transcriptional regulation
Sp1
N terminus
Transcriptional regulation
Gln-Ala repeat transcriptional activator (CA150) CIP4
Full-length huntingtin
Transcriptional regulation
N terminus
NF-kB/Rel/dorsal family transcription factor HAP1
C terminus
Involved in Cdc42 and WASp-dependent signal transduction Nuclear transport
HIP1
N terminus
HIP1-related/HIP12
Does not interact with huntingtin but can interact with HIP1
HIP14
N terminus
Hippi
Binds to HIP1
PACSIN I/syndapin,
N terminus, proline-rich region
Endophilins
Exon 1 protein, proline-rich region
PSD-95
N-terminal proline region
N terminus
Endosome – lysosome trafficking Clathrin-mediated endocytosis via binding to clathrin and AP2; promotes clathrin assembly; does not bind to actin Acts as functional link between clathrin and actin; promotes actin organization and clathrin assembly Intracellular trafficking and endocytosis Mediates apoptotic pathways.
Binds to dynamin, synaptojanin and N-WASp Bind to lipids; acts as a lysophosphatidic acid acyl transferase; interacts with dynamin and amphiphysins Binds and regulates the activity of glutamate receptors
[63]
Enhanced by lengthening of the adjacent glutamine tract Depleted from nucleus and present in aggregates; decreased transcription Enhanced interaction; disrupted interaction between Sp1 and TAFII130 Marked increase of CA150 expression Marked CIP4 overexpression and cell death induction of striatal neurons ?
[64]
[70]
Enhanced binding
[71]
Reduced interaction
[72 –74]
[46,65,66]
[51,67]
[68] [69]
[74,75]
Decreased interaction
[49]
Free HIP1 modulated by polyglutamine length in huntingtin; forms Hippi – HIP1 heterodimers and launches apoptosis Enhanced interaction
[76]
Enhanced binding to endophilin A3
[57,78]
Decreased interaction
[56]
[33,58,77]
a
Abbreviations: CA150, co-activator 150; CAG, Cys-Ala-Gly; CBP, CREB-binding protein; CIP4, cdc42-interacting protein 4; CREB, cAMP-response-element-binding protein; HAP1, Huntingtin-associated protein 1; HIP1, Huntingtin-interacting protein 1; HIP14, Huntingtin-interacting protein 14; Hippi, HIP1 protein interactor; htt, huntingtin; HYPA, htt yeast partner A; HYPB, htt yeast partner B; HYPC, htt yeast partner C; N-CoR, nuclear receptor co-repressor; PXDLS, Pro-Xaa-Asp-Leu-Ser; Sp1, specificity protein 1; TAF, TATA-binding protein-associated factor; WW, Trp-Trp.
endocytic and/or autophagic pathway and synaptic proteins that would be digested by this proteolytic pathway as part of the normal turnover of cellular protein [14,15]. Mutant huntingtin and synaptic dysfunction Distinct morphological abnormalities in neuronal dendrites are apparent as early degenerative changes both in transgenic mouse models of HD and in individuals affected with HD [16]. A marked decrease in the number of dendritic spines and a thickening of proximal dendrites has been observed before cell death is detectable [17]. Clinically, some individuals with genetically confirmed HD have been reported to develop severe motor and cognitive deficits and mental deterioration, even in the absence of widespread neuronal cell loss, as assessed by postmortem http://tmm.trends.com
examination of their brains [4]. Recent imaging studies have shown that there is progressive striatal and cortical dysfunction of the dopamine D2 receptor in individuals with HD [18]. Mutant huntingtin has been shown to impair directly the cellular machinery involved in synaptic transmission. Much insight has been gained by studies in the R6/1 and R6/2 transgenic mouse models of HD. These mice express exon 1 of the gene encoding human huntingtin containing a CAG repeat expansion of 115 or 150, respectively [19]. Corticostriatal glutamatergic fibers represent the principal excitatory input to the striatum [20]. Cellular pathology caused by mutant huntingtin in the presynaptic terminals might result in an increased release of glutamate. Alternatively, impaired clearance of glutamate
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from the synaptic cleft might increase glutamatergic neurotransmission. In both cases, striatal excitotoxicity could occur [21,22]. Intracerebral microdialysis has shown that depolarizing concentrations of potassium chloride increase the extracellular concentrations of glutamate substantially more in R6/1 mice than in wild-type mice [23] and, in particular, the glial glutamate transporter (GLT-1) is downregulated in R6/2 mice before any evidence of neurodegeneration [24]. This indicates that there is an impairment in glutamate transport and glutamate –glutamine cycling, and suggests that a defect in astrocytic glutamate uptake could contribute to the phenotype and to neuronal cell death in HD. Furthermore, mutant huntingtin binds to synaptic vesicles with higher affinity than does the wildtype form and inhibits the uptake of glutamate in synaptic vesicles in a dose-dependent manner [25]. With regard to the dopaminergic input to the striatum, intracerebral microdialysis experiments have detected substantially decreased concentrations of striatal extracellular dopamine in 16-week-old R6/1 mice [26]. Notably, this occurs at an age when there is no neuronal death in the substantia nigra or in the striatum. Moreover, postmortem tissue amounts of dopamine are unchanged in R6/1 mice at this age, indicating that their capacity to synthesize dopamine is still normal and suggesting that their synaptic function is perturbed [26]. Similarly, the striatal cholinergic system is also affected by mutant huntingtin in mouse models of HD. The evoked release of acetylcholine, its M2 autoreceptormediated maximum inhibition and its dopamine D2 heteroreceptor-mediated maximum inhibition are all substantially diminished in R6/2 mice as compared with wild-type mice [27]. In addition, the activity of choline acetyltransferase and the uptake of synaptosomal highaffinity choline decrease progressively with age [27]. R6/2 mice also show a significant reduction in long-term potentiation and long-term depression in the hippocampus [28]. In a different mouse model of HD, excitatory synapses in the hippocampus are impaired in their ability to sustain transmission during repetitive stimulation and there is indirect evidence that glutamate release is significantly reduced during higher frequency synaptic activation as compared with controls [29]. Taken together, there is much evidence for deficits that are located on the presynaptic side of the synapse in the machinery that regulates the release of neurotransmitters. How can mutant huntingtin impair normal neurotransmission? The molecular mechanisms underlying impaired neurotransmission in HD are still largely unknown. However, recent studies have provided important clues by showing that several synaptic vesicle proteins that are essential for synaptic transmission are abnormal in individuals with HD and HD transgenic mice [30 – 33]. Normal synaptic transmission is regulated by a large group of synaptic vesicle proteins that have key roles in membrane trafficking in exocytotic or endocytotic processes [34,35]. Alterations in the expression, post-translational modification and patterns of protein – protein interaction http://tmm.trends.com
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of these proteins have important implications for the function of neurons. Progressive loss of neuronal complexin II, a protein that regulates fusion processes between synaptic vesicles and the plasma membrane, has been shown in cells expressing mutant huntingtin, in R6/2 transgenic mice and in the brains of individuals with HD [30,31]. Furthermore, overexpression of complexin II in cultured cells expressing mutant huntingtin rescues the cells from premature death [36]. Additional evidence for changes in synaptic function in mouse models of HD comes from observations of an abnormal phosphorylation state of synapsin I in the striatum and the cerebral cortex of R6/2 transgenic mice. Such changes do not result from modifications at the level of protein expression [32], however, which suggests that an early impairment in synapsin phosphorylation/ dephosphorylation might alter synaptic vesicle trafficking and lead to defective neurotransmission in HD. Furthermore, PACSIN 1/syndapin, a neurospecific phosphoprotein with a central role in synaptic vesicle and receptor recycling, interacts with huntingtin. This interaction is dependent on the length of the polyglutamine repeat and is enhanced by the huntingtin mutation. In the presence of mutant huntingtin, PACSIN 1 seems to be removed from synapses and neuronal processes [33]. Several lines of mice with inactivated genes encoding synaptic vesicle proteins and/or huntingtin-interacting proteins show neuropathological changes. Among these, HIP1 knockout mice develop tremor and gait ataxia. The mice show a decreased assembly of endocytic protein complexes and profound defects in the internalization of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors containing glutamate receptor 1 (GluR1) [37]. In Drosophila, an absence of endophilin markedly impairs endocytosis in neuromuscular junctions and in synapses in the central nervous system [38,39]. Deletion of complexin II has been observed in different models of HD [30,31,36]. It has been shown that, although mice deficient for complexin II show no obvious neurological changes, long-term potentiation in both the CA1 and the CA3 regions of the hippocampus is impaired [40]. By contrast, homozygous mice deficient for complexin I develop severe gait ataxia, suffer from sporadic seizures and die 2– 4 months after birth, also in the absence of any obvious morphological brain changes [41]. Mice lacking both complexin I and complexin II die perinatally, even though there are no changes in brain cytoarchitecture [41]. These observations further strengthen the hypothesis that the dysfunction of synaptic vesicle proteins might be important in early events associated with HD. How does mutant huntingtin lead to an abnormality of synaptic vesicle proteins in HD? The mechanisms underlying changes in synaptic function caused by the presence of an expanded polyglutamine protein are still not well understood. As mentioned above, the expression of mutant huntingtin might lead to a general disruption of protein function, either by recruiting proteins to aggregates or by causing functional inhibition of the ubiquitin– proteasome system. Mutant huntingtin might also directly cause changes in protein expression.
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Microarray studies have shown that the patterns of changes in gene expression are complex in both cell and animal models of HD [42,43]. Although these patterns are not completely consistent among the different models, they show that mutant huntingtin can profoundly affect gene expression [44]. Huntingtin binds and interacts with several key transcription factors or coactivators (Table 1). There is evidence that the expanded polyglutamine stretch is crucial to its interaction with transcription factors such as TAFII130, a coactivator involved in transcriptional activation dependent on the cAMP-responsive element (CRE)-binding protein (CREB). The expanded polyglutamine stretch strongly suppresses CREB-dependent transcriptional activation [45], as well as depleting CREB-binding protein (CBP) from nuclear locations and specifically interfering with CBP-activated gene transcription [46]. Thus, mutant huntingtin can disrupt gene transcription through aberrant interactions with transcriptional factors. Moreover, some transcription factors need to be processed by the proteasome before they are activated [47]. Thus, there is more than one pathway by which mutant huntingtin could compromise the transcription of specific synaptic vesicle proteins, among which the expression of synaptoporin, N-ethylmaleimide sensitive factor (NSF) and synaptobrevin I is known to be markedly attenuated in HD (Figure 1) [42,43]. An alternative explanation for the disruption of synaptic function by mutant huntingtin is that the expanded polyglutamine stretch alters the interactions of wild-type huntingtin with other key proteins. This model can be viewed as a partial loss of normal function of the wild-type protein, leading to a disruption of the manner in which synaptic proteins function (Figures 1 and 2). After a (a) Normal Gene transcription
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protracted period (several years in humans) of failed synaptic protein function, cell death is eventually triggered. Botulinum toxins are known to disrupt synaptic function by cleaving the soluble NSF attachment protein (SNAP) receptor (SNARE) complex and, notably, this synaptic dysfunction can cause the subsequent degeneration of axons, dendrites and cell bodies of neurons, thereby leading to cell death [48]. Importantly, wild-type huntingtin interacts with several vesicle proteins that are vital for endocytosis. For mutant huntingtin, these interactions might be impaired and consequently might affect endocytic processes. For example, the binding affinity of huntingtin to PACSIN 1 is dependent on the length of the polyglutamine tract [33]; therefore, an increased interaction between the two proteins could lead to a sequestration of PACSIN 1 into non-synaptic aggregates. In addition, aberrant protein interactions could conceivably occur between pre-fibrillar species of huntingtin, which might be important in defective synaptic transmission. HIP14, another huntingtininteracting protein, has a role in intracellular transport processes and endocytosis [49]. A decreased interaction between mutant huntingtin and HIP14 in HD can impair endocytosis [49]. In analogy to synaptic vesicles, autophagic vacuoles also rely on the recycling of membranes through the formation of endosomes, and this process might be affected in HD. Thus, under conditions of increased oxidative stress, autophagic vacuoles accumulate excessively in the cytoplasm of R6/2 striatal neurons [15,50], indicating either an abnormality in the primary response to oxidative stress or a disturbance of vesicle cycling. Aggregates containing mutant huntingtin are usually located in the nucleus and cytoplasm, but they can also
(b) Huntington's disease Gene transcription –
Synaptic proteins
–
Synaptic proteins
Mutant huntingtin
+ or – Protein interaction
Protein interaction
Activation of cell death program
Transmitter release, synaptic vesicle cycling
Altered transmitter release and receptors
Excitotoxicity
Neurotransmission (including LTD/LTD)
Impaired neurotransmission
Neurodegeneration
Normal brain function
Motor symptoms, cognitive impairments, psychiatric disturbances TRENDS in Molecular Medicine
Figure 1. Principal cellular processes involved in the regulation of synaptic transmission. (a) Regulation under normal conditions. (b) Regulation under the pathological influences of mutant huntingtin. The most important take-home message is that not only does mutant huntingtin cause cell death directly, but also it can impair synaptic transmission at several levels, thereby leading to symptoms. http://tmm.trends.com
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Endocytosis
Exocytosis Actin
*
* Synapsin I
Synaptotagmin Synaptobrevin
HIP1R
N-WASp
HIP1
Rabphilin 3A
Clathrin-associated proteins
PACSIN 1 Rab3a
Complexin II
Endophilins Abp1p
Dynamin
SNAP-25
Syntaxin TRENDS in Molecular Medicine
Figure 2. Protein–protein interactions among key factors involved in exocytosis and endocytosis. Red arrows between individual proteins indicate specific binding interactions between those components. Dashed blue boxes indicate protein–protein interactions that potentially could be altered by mutant huntingtin, thereby disturbing endocytosis and exocytosis. Abbreviations: HIP1, Huntingtin-interacting protein 1; HIP1R, HIP1-related protein; N-WASp, neuronal Wiskott– Aldrich syndrome protein; PACSIN 1, protein kinase C and casein kinase in neurons 1.
appear in the axon and nerve terminals [25]. As discussed above, mutant huntingtin binds to synaptic vesicles with higher affinity than does wild-type huntingtin and inhibits the uptake of glutamate by synaptic vesicles [25], suggesting that it might be able to affect synaptic homeostasis directly. Presynaptic versus postsynaptic involvement In the above sections, we focused on the role of presynaptic mechanisms in the pathogenesis of HD symptoms. But are postsynaptic mechanisms also important in this process? Mutant huntingtin can downregulate several Sp1-dependent neuronal genes, including those encoding the dopamine D1, D2 and D3 receptors that are localized in both pre- and postsynaptic components [51]. In HD, mutant huntingtin selectively decreases the expression of N-methyl-D-aspartic acid (NMDA) receptors at presymptomatic stages [52]. At later stages, binding to individual ionotropic glutamate receptors is decreased and there is a less marked reduction in metabotropic glutamate receptor binding. In addition, there is a selective loss of kainic acid and AMPA binding in the frontal cortex [53]. In medium-sized spiny neurons of R6/2 HD transgenic mice, currents induced by the selective activation of NMDA receptors are enhanced, cytoplasmic Ca2þ concentration is increased and the frequency of spontaneous excitatory postsynaptic currents is raised [54,55]. Furthermore, wild-type huntingtin is associated with NMDA and kainate receptors through postsynaptic density 95 (PDS-95), whose Src homology domain 3 mediates the binding to huntingtin [56]. Thus, huntingtin could be involved in signal transduction immediately downstream of the neurotransmitter receptors. Notably, expanded polyglutamine stretches interfere with the ability of huntingtin to interact with PSD-95 and, in http://tmm.trends.com
turn, influence the normal function of NMDA and kainate receptors [56]. In addition, normal receptor recycling in nerve terminals is regulated by several proteins that also bind to huntingtin [33,57]. Proteins such as PACSIN 1 and endophilins are thought to participate in the regulation of receptor internalization by linking the actin polymerization machinery to endocytic protein complexes [58,59]. Mutant huntingtin might also impair proper receptor recycling in postsynaptic neurons. Finally, it is worth mentioning that selective reduction of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) has been observed in presymptomatic mouse models of HD [60]. DARPP-32 has a key role in many neurotransmitter pathways [61] and is involved in controlling receptors and ion channels, among others. DARPP-32 is involved not only in dopamine signal transduction, but also in signaling via many other neurotransmitters, including glutamate and serotonin. Therefore, a reduction of DARPP-32 in HD might contribute to the dysfunction of NMDA, AMPA and dopamine receptors and might disrupt normal synaptic transmission. Concluding remarks and future directions Little is known about the normal cellular function of huntingtin or how its function is altered by an expansion of polyglutamine. Recent studies suggest that the polyglutamine expansion might enable mutant huntingtin to corrupt normal gene transcription [51] and to disrupt normal intracellular trafficking and transmitter release [25,54]. But several issues remain with regard to how cellular dysfunction, including synaptic disturbances, can lead to neurodegeneration. Molecular genetic approaches will be useful for dissecting the roles of various factors in HD. Improved understanding of such interactions will pave the way for
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targeting one or more of the aberrant protein – protein interactions in novel therapies. Eventually, it might be feasible to develop drugs that alleviate the negative effect of polyglutamine expansions by interfering with specific pathways of gene expression or by altering undesirable protein – protein interactions. This could allow intervention in the disease process very much upstream of the events that eventually lead to cell death. The idea that we might be able to inhibit events in the pathogenetic cascade early on raises the exiting possibility that ultimately the development of clinical symptoms might be completely prevented. Note added in proof Very recently, it was demonstrated that nerve terminals in different HD animal models have far fewer synaptic vesicles [79]. Mutant huntingtin binds more tightly to synaptic vesicles compared with wild-type huntingtin; glutamate release is substantially reduced in the HD brain slice. These observations indicate impairment of synaptic function in HD.
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Acknowledgements ˚ sa Peterse´n and Ruben Smith for critically reading the We thank A manuscript. Work cited from our laboratories is supported by grants from the Swedish Research Council, the Hereditary Disease Foundation, the Swedish Society for Medicine, Crafoord Foundation and Hedlund Foundation, the Center for Molecular Medicine (CMMC; TP78) and Ko¨ln Fortune from the Medical Faculty of the University of Cologne. Some of the ideas presented in this article were stimulated by discussions in European Research Project (Concerted Action) entitled Early Pathogenetic Markers Of Slow Neurodegenerative Diseases (EPSND).
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