Neuroprotective Effects of σ2R/TMEM97 Receptor Modulators in the Neuronal Model of Huntington's Disease

Abstract

Huntington's disease (HD) is a genetic neurodegenerative disease caused by an expanded CAG repeat in the Huntingtin (HTT) gene that encodes for an expanded polyglutamine (polyQ) repeat in exon-1 of the human mutant huntingtin (mHTT) protein. The presence of this polyQ repeat results in neuronal degeneration, for which there is no cure or treatment that modifies disease progression. In previous studies, we have shown that small molecules that bind selectively to σ₂R/TMEM97 can have significant neuroprotective effects in models of Alzheimer's disease, traumatic brain injury, and several other neurodegenerative diseases. In the present work, we extend these investigations and show that certain σ₂R/TMEM97-selective ligands decrease mHTT-induced neuronal toxicity. We first synthesized a set of compounds designed to bind to σ₂R/TMEM97 and determined their binding profiles (K_i values) for σ₂R/TMEM97 and other proteins in the central nervous system. Modulators with high affinity and selectivity for σ₂R/TMEM97 were then tested in our HD cell model. Primary cortical neurons were cultured in vitro for 7 days and then co-transfected with either a normal HTT construct (Htt N-586-22Q/GFP) or the mHTT construct Htt-N586-82Q/GFP. Transfected neurons were treated with either σ₂R/TMEM97 or σ₁R modulators for 48 h. After treatment, neurons were fixed and stained with Hoechst, and condensed nuclei were quantified to assess cell death in the transfected neurons. Significantly, σ₂R/TMEM97 modulators reduce the neuronal toxicity induced by mHTT, and their neuroprotective effects are not blocked by NE-100, a selective σ₁R antagonist known to block neuroprotection by σ₁R ligands. These results indicate for the first time that σ₂R/TMEM97 modulators can protect neurons from mHTT-induced neuronal toxicity, suggesting that targeting σ₂R/TMEM97 may lead to a novel therapeutic approach to treat patients with HD.

Keywords: Huntington’s disease; neuronal survival; neuroprotection; nucleus condensation; σ2R/TMEM97.

Figure 1.. Structure of σ₂R/TMEM97-selective modulators and their binding affinities.

A. Structures of racemic B-norbenzomorphans (AMA-1127 and DKR-1677) and methanobenzazocines (DKR-1051 and UKH-1114) that are selective modulators of σ₂R/TMEM97 and their binding affinities. K_i, for σ₂R/TMEM97 (rat) and σ₁R (guinea pig). B. Structures of racemic B-norbenzomorphans (JJS-1678, BJM-1679, and EES-1686) and methanobenzazocines (BEA-1687) that are selective modulators of σ₂R/TMEM97 and their binding affinities. K_i, for σ₂R/TMEM97 (human) and σ₁R (human). C. Structures of σ₁R-selective modulators MPC-1154 and racemic HLJ-1560 and their binding affinities, K_i, for σ₂R/TMEM97 (rat) and σ₁R (guinea pig).

Figure 2.. Neuroprotective effect of σ₂R/TMEM97-selective modulators on mHTT induced toxicity.

Primary cortical neurons were co-transfected with either Htt-N586–22Q or Htt-N586–82Q and GFP. Four hours after transfection, neurons were treated with either σ₁R or σ₂R/TMEM97 modulators for 48 h, whereupon neurons were fixed, and nuclei were stained with Hoechst. Cells with condensed nuclei were counted as dead cells. Only neurons transfected with plasmid were counted. A. Representative pictures for neurons transfected with Htt-N586–22Q or Htt-N586–82Q and GFP. B. Representative pictures for neurons transfected with Htt-N586–82Q/GFP and treated with σ₂R/TMEM97 or σ₁R modulators. Neurons were treated with 1 μM of the indicated modulators. Insert boxes indicated viable cells with normal nucleic morphology. C-H. σ₂R/TMEM97-selective modulators tested in HD cell model showing neuroprotection. These compounds are AMA-1127 (C), DKR-1051 (D), UKH-1114 (E), BJM-1679 (F), EES-1686 (G), and BEA-1687 (H). ^### p<0.0001 vs Htt N586–22Q with 0. * p<0.05, *** p<0.0001 vs Htt N586–82Q with 0. n=6–8 independent experiments.

Figure 3.. Neuroprotection effect of σ₁R-selective modulators on mHTT induced toxicity.

Primary cortical neurons were co-transfected with either Htt-N586–22Q or Htt-N586–82Q and GFP. Four hours after transfection, neurons were treated with or without σ₁R modulators at different concentrations. σ₁R-selective modulators tested in HD cell model were as follows: MPC-1154 (A) or HLJ-1560 (B). ^### p<0.0001 vs Htt N586–22Q with 0. *** p<0.0001 vs Htt N586–82Qwith 0. n=6–8 independent experiments.

Figure 4.. Specificity of σ₂R/TMEM97 modulators.

To further explore the specificity of σ₂R/TMEM97 modulators, selective σ₁R antagonist, NE-100 was used in the primary cortical neurons treated σ₁R or σ₂R/TMEM97 modulators (10 μM). Primary cortical neurons were co-transfected with either Htt-N586–22Q or Htt-N586–82Q and GFP. Four hours after transfection, neurons were treated with modulators with or without a pretreatment with 10 μM of NE-100. After 48 h, neurons were fixed and nuclei were stained. We included one σ₁R-selective modulator, HLJ-1560, as a positive control, which its effect will be blocked by NE-100. Cell death were quantified using a nuclei condensation assay. NE-100 abolished the protective effect of the σ₁R modulator, HLJ-1560, but it did not influence the effects of σ₂R/TMEM97 modulators. *** p<0,0001 vs Htt N586–82Q, # p<0,001 vs Htt N586–22Q. n=4–6 independent experiments.

Scheme 1.

Synthesis of AMA-1127

Scheme 2.

Syntheses of JJS-1678 and EES-1686

Scheme 3.

Synthesis of BJM-1679

Scheme 4.

Synthesis of BEA-1687.

Scheme 5.

Synthesis of HLJ-1560.