Presynaptic Dysfunction

4.1. Axonal Transport

The discovery that htt microaggregates form preferentially in the neuropil of MSNs, and especially in their projection target areas, in knock-in HD mice (HdhQ) that do not develop an overt HD phenotype suggested that MSN axonal dysfunction is one of the earliest pathological changes in HD8. These aggregates also formed in mhtt-expressing cultured striatal cells where they blocked protein transport and caused neuritic degeneration8. Following this report, two studies added support to the axonal defect theory for HD pathogenesis, convincingly demonstrating that htt is required for normal axonal transport, and that expanded polyQ-containing peptides produce severe axonal transport deficits in Drosophila9 and squid axoplasm10. Recent work has produced similar findings in mammalian neurons in vivo and in vitro11. Data from earlier subcellular localization studies also support the idea that axonal dysfunction may contribute to HD pathogenesis4.

The first htt-interacting protein described, Huntingtin-Associated Protein 1 (HAP1), is highly expressed in the brain and binds htt in a manner that is enhanced by expanded polyQ12. Like htt, HAP1 localizes with membrane-bound organelles (including vesicles), dendritic and axonal microtubules13, and the vesicular fractions of nerve terminals, where it interacts with the p150 subunit of dynactin1415, a protein required by the vesicular microtubule transport protein dynein. Perturbation of htt/HAP1-dependent vesicular trafficking by polyQ expansion may underlie the decreased synaptic vesicle number and HAP1-vesicle interactions, as well as impaired neurotransmitter (NT) release, observed after phenotypic onset in two HD mouse models16. Axonal transport of the MSN prosurvival factor, brain-derived neurotrophic factor (BDNF), is quickened by htt via its association with HAP1 and is much reduced by mhtt, resulting in neurotoxicity15. These data and others suggest that htt-HAP1 interactions are disrupted by polyQ expansion, causing a deficit in BDNF trophic support from the cortex to the striatum and the development of striatal neurodegeneration14.

4.2. Vesicle Fusion and Neurotransmitter Release

Electrophysiological investigation of hippocampal synaptic plasticity in HD knock-in mice (HdhQ) demonstrated impaired NT release during periods of sustained activity17. Subsequent investigations have shown, at the cortico-striatal synapse of R6/2 mice, that unusually frequent large-amplitude synaptic events occur, with a concomitant and progressive reduction in the frequency of miniature excitatory postsynaptic currents (mEPSCs), beginning at the same time as the behavioral phenotype18. Together such observations suggest that mhtt interferes directly with the vesicle release cycle.

The synapsin family of proteins associates with synaptic vesicles and elements of the cytoskeleton and, although the exact function of synapsins remains unclear, this binding may regulate the size of the synaptic vesicle reserve pool (and concomitantly the readily releasable pool; RRP) at axon terminals (reviewed in ref. 19). Phosphorylation of synapsin 1 reduces its affinity for binding both vesicles and F-actin; and although synapsin 1 expression is unaltered in R6/2 HD mice20,21, the levels of phosphorylated synapsin 1 are increased after onset of motor symptoms20. This suggests that, at this age, R6/2 mice may exhibit alterations in the size of the RRP. These data may help explain the unusually large spontaneous EPSCs observed at the cortico-striatal synapse around this time18. Synapsin 1 also interacts with the synaptic vesicle-associated small G protein Rab3A, which appears to negatively regulate a late step of synaptic vesicle fusion, controlling quantal release (reviewed in ref. 19). Thus, synapsin hyperphosphorylation20 may also disrupt vesicular release and contribute to the reduced mEPSC frequency in R6/2 mice after the onset of symptoms18. More recent analysis of mRNA and protein levels of a number of synaptic proteins in the less severe phenotype of the R6/1 mouse has demonstrated that a Rab3A-interacting protein, Rabphilin 3A, is selectively reduced in cortex at the age at which symptoms appear22.

Levels of the synaptic vesicle release protein complexin II (CPLXII) are selectively and progressively depleted in R6/2 mice23 and human HD tissue24. Furthermore, PC12 cells expressing mhtt also show reduced CPLXII with concomitantly reduced exocytosis that was reversed by CPLXII overexpression25. Importantly, CPLXII mRNA expression is also reduced in individual striatal cells of R6/2 mice, prior to the onset of overt phenotype26 and temporally associated with the appearance of cognitive deficits27. Analysis of CPLXI/II knock-out neurons suggests that complexins positively regulate synaptic vesicle fusion28, consistent with a reduction of complexin II underlying reduced exocytosis in PC12


Mice lacking complexin II have near-normal basal hippocampal transmission but reduced short-term potentiation and long-term potentiation (LTP)29, suggesting that NT release may be impaired during periods of sustained activity. These observations are strikingly similar to those from R6/2 mice30. Together, these studies suggest that reduced LTP and cognitive deficits in the R6/2 may be caused, at least in part, by altered CPLXII function. However, in the presence of the NMDA receptor antagonist AP5, short-term potentiation in R6/2 mice is similar to controls, suggesting that altered NMDAR activity, and not presynaptic NT release per se, is causal to differences in short-term potentiation30. Moreover, while complexin II knock-out mice do exhibit progressive cognitive and motor deficits31, which are similar to those seen in R6/2 mice, the phenotype is mild in comparison to R6/2 and occurs at a later age. Thus, R6/2 cognitive and motor deficits may be only partially attributable to reduced CPLXII expression.

Other proteins that regulate NT release at the presynaptic terminal are also implicated in HD. Expanded mhtt, but not htt, binds the Cysteine String Protein (CSP), a molecular chaperone expressed on synaptic vesicles, and blocks its tonic inhibition of N-type Ca2+ channels32. NT release is also modulated by presynaptic metabotropic glutamate (mGluRs), NMDA, cannabinoid and dopamine receptors (DARs), all of which are present on cortical terminals and altered in HD (see Section 5).

4.3. Vesicle Recovery

The huntingtin-interacting protein 1 (HIP1) binds and colocalizes with the N-terminal portion of huntingtin33,34, and the efficacy of huntingtin/HIP1 binding is diminished by polyglutamine expansion34. HIP1 also binds clathrin and is thus implicated in clathrin-mediated endocytosis, a process that facilitates the rapid retrieval of synaptic vesicles at the presynaptic terminal35.

The disruption of this and other interactions (e.g., see Section 4.1) by polyglutamine expansion is evidence for "loss of function" effects relating to NT release. In addition, "gain of function" effects may also be important in the HD vesicle cycle. For example, htt binding of the protein kinase C and casein kinase substrate in neurons protein 1 (PACSIN1: a regulator of vesicle recovery) is increased proportionally to polyQ repeat length36, and PACSIN1 appears to be abnormally distributed away from synaptic compartments in HD tissue36.

4.4. Glutamate Uptake

In addition to disruption of vesicular release of NT, synaptic transmission may also be perturbed at the HD synapse by abnormal clearance of glutamate from the synaptic cleft. In situ hybridization studies of astrocytic glutamate transporter (EAAT1/GLT1) mRNA in the neostriatum of post mortem HD brains revealed decreased GLT1 message that was related to reduced expression within astrocytes37. GLT1 mRNA and glutamate uptake are also reduced in R6/1 and R6/2 mice after phenotypic onset20,38. Such dysfunction may underlie the increases in basal hippocampal transmission that are observed in R6/1 mice long after the onset of phenotype (A. Milnerwood, in press).

4.5. Presynaptic Dysfunction and HD Symptom Progression

Several lines of evidence detailed above provide mechanisms by which glutamate release might be perturbed by mhtt. Consistent with these findings, a variety of studies have suggested that progressively reduced presynaptic function occurs at or after the onset of overt symptoms in high polyQ, N-terminal fragment models16,18,39, that is not observed at early stages in full-length construct YAC72 mice40. In light of these changes, it is interesting that a combination drug therapy aimed at increasing NT levels in R6/2 mice delayed cognitive impairment and some alterations in gene expression, in addition to producing a 7% increase in survival; however, motor symptoms continue unabated41.

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