Huntingtin-interacting protein 1 influences worm and mouse presynaptic function and protects Caenorhabditis elegans neurons against mutant polyglutamine toxicity

Abstract

Huntingtin-interacting protein 1 (HIP1) was identified through its interaction with htt (huntingtin), the Huntington's disease (HD) protein. HIP1 is an endocytic protein that influences transport and function of AMPA and NMDA receptors in the brain. However, little is known about its contribution to neuronal dysfunction in HD. We report that the Caenorhabditis elegans HIP1 homolog hipr-1 modulates presynaptic activity and the abundance of synaptobrevin, a protein involved in synaptic vesicle fusion. Presynaptic function was also altered in hippocampal brain slices of HIP1-/- mice demonstrating delayed recovery from synaptic depression and a reduction in paired-pulse facilitation, a form of presynaptic plasticity. Interestingly, neuronal dysfunction in transgenic nematodes expressing mutant N-terminal huntingtin was specifically enhanced by hipr-1 loss of function. A similar effect was observed with several other mutant proteins that are expressed at the synapse and involved in endocytosis, such as unc-11/AP180, unc-26/synaptojanin, and unc-57/endophilin. Thus, HIP1 is involved in presynaptic nerve terminal activity and modulation of mutant polyglutamine-induced neuronal dysfunction. Moreover, synaptic proteins involved in endocytosis may protect neurons against amino acid homopolymer expansion.

Figure 1.

Molecular characterization of hipr-1. A, Sequence alignment (using ClustalW) of human HIP1 and the C. elegans homolog HIPR-1. Identical amino acids are highlighted in black, with similar amino acids in gray. Overall, there is 32% identity and 49% similarity between C. elegans HIPR-1 and human HIP1. B, The hipr-1 insertion/deletion allele tm2207 was obtained by PCR screening of a UV-TMP mutagenesis library. The Coils program was used to predict the probability of coiled-coil domains (Lupas et al., 1991).

Figure 2.

Phenotypic characterization of hipr-1 mutants. A, Defecation cycle times were lengthened in hipr-1(tm2207) mutants. The mean time interval between consecutive posterior body muscle contractions was scored. *p < 0.01 versus wild type; **p < 0.001 versus RNAi controls; ***p < 0.001 versus wild type. Error bars indicate SEM (n > 20). B, Average brood size was reduced by mutation or RNAi of hipr-1 (n > 30). *p < 0.001 compared with wild type; **p < 0.001 versus RNAi controls; ***p < 0.001 versus wild type. Error bars indicate SEM.

Figure 3.

hipr-1 regulates synaptic activity and localization of synaptobrevin. A, Cholinergic neuronal transmission was measured by determining the onset of paralysis induced by the cholinesterase inhibitor aldicarb. hipr-1(tm2207) mutants were resistant to the paralytic effects of aldicarb. Strains unc-31, unc-64, and unc-29 are deficient for CAPS, syntaxin, and a non-α-subunit of the nicotinic acetylcholine receptor, respectively. Data points are the mean and SEM for at least five independent experiments. *Significantly different from wild type (p < 0.001). B, hipr-1 mutants were sensitive to paralysis by the nicotinic acetylcholine agonist levamisole. unc-29 mutants were resistant to the paralytic effects levamisole, because it acts on receptors in body wall muscle. *p < 0.01, **p < 0.001 versus wild type. C, SNB-1::GFP expression in the ventral cord for jsIs37 and hipr-1(tm2207);jsIs37 animals. Scale bar, 5 μm.

Figure 4.

Recovery from synaptic depression is delayed in slices from HIP1^−/− mice. A, HIP1^−/− neurons require more time to recover from synaptic depression in comparison with wild-type littermate controls. CA1 pyramidal cells were voltage clamped at −60 mV and stimulated every 3 s via Schaffer collaterals to obtain baseline synaptic responses in the presence of AP5. A prolonged tetanus (10 Hz, 200 s; bar) was delivered at time 0 to induce synaptic depression including depletion of the readily releasable synaptic vesicle pool. Recovery from depression was studied by recording EPSCs at the original stimulation rate (data were averaged from n = 9 wild-type and n = 6 HIP1^−/− neurons). B, Same data as in A were plotted on an expanded time base to compare the initial potentiation of EPSCs during the tetanus demonstrating enhanced facilitation in HIP1^−/− mice (mean ± SEM). C, The recovery after synaptic depression is significantly decreased in HIP1^−/− neurons in comparison with wild-type neurons. The same data as in A were replotted to show only the synaptic responses at the end of the tetanus and most of the recovery phase. Each point represents six averaged EPSCs ± SEM (t test; *p < 0.05).

Figure 5.

Short-term plasticity is enhanced in HIP1^−/− mice. A, Sample fEPSP traces evoked by delivering paired pulses 40 or 60 ms apart (only the second response is shown for the latter) to CA3–CA1 synapses from wild-type (blue lines; top traces) and HIP1^−/− mice (red lines; middle traces). Traces represent average from three hippocampal slices per genotype selected for similar average initial fEPSP slope. Traces have been superimposed in the bottom panel for comparison. ACSF contained 2.0:2.0 mm ratio of Ca²⁺/Mg²⁺. B, PPF is significantly increased in HIP1^−/− neurons (n = 18) compared with wild-type littermate controls (n = 17). Interpulse intervals between paired pulses were varied between 40 and 600 ms. Each measurement represents the mean ± SEM (t test, *p < 0.04). PPF is elevated but no longer significantly different in HIP1^−/− neurons (n = 20) under increased release probability conditions (Ca²⁺/Mg²⁺ ratio was increased to 2.5:1.3) in comparison with wild-type neurons (n = 17; mean ± SEM).

Figure 6.

hipr-1 protects against mutant polyQ neuronal dysfunction. A, Mutation or RNAi of hipr-1 decreased touch responsiveness in animals expressing mutant htt. *p < 0.001 compared with 19Q alone; **p < 0.001 versus 128Q alone; ***p < 0.001 versus 128Q RNAi controls (n = 200 for all experiments). B, In vivo analysis revealed no change in the frequency of either the aggregation of huntingtin fusion proteins or dystrophy along axonal processes (n = 200). C, Synaptic genes are required for wild-type touch response. The percentage of wild-type response is given for each LOF synaptic mutant. *p < 0.001 compared with wild type (n = 200). D, Synaptic genes associated with endocytosis protect against mutant polyQ neuronal dysfunction. The percentages of either 128Q or 19Q touch response for each LOF mutant are given. *p < 0.001 versus 128Q animals; **p < 0.001 compared with 19Q controls (n = 200 for all experiments).