Chronic administration of cholesterol oximes in mice increases transcription of cytoprotective genes and improves transcriptome alterations induced by alpha-synuclein overexpression in nigrostriatal dopaminergic neurons

Highlights

• Cholesterol-oximes increase stress defense mRNAs in nigrostriatal dopaminergic neurons.

• Cholesterol-oximes increase dopamine-related mRNAs in nigrostriatal dopaminergic neurons.

• Alpha-synuclein over-expression induces transcriptome changes in nigrostriatal neurons.

• Cholesterol-oximes reverse a subset of these alpha-synuclein induced alterations.

Abstract

Cholesterol-oximes TRO19622 and TRO40303 target outer mitochondrial membrane proteins and have beneficial effects in preclinical models of neurodegenerative diseases leading to their advancement to clinical trials. Dopaminergic neurons degenerate in Parkinson's disease (PD) and are prone to oxidative stress and mitochondrial dysfunction. In order to provide insights into the neuroprotective potential of TRO19622 and TRO40303 for dopaminergic neurons in vivo, we assessed their effects on gene expression in laser captured nigrostriatal dopaminergic neurons of wildtype mice and of mice that over-express alpha-synuclein, a protein involved in both familial and sporadic forms of PD (Thy1-aSyn mice). Young mice were fed the drugs in food pellets or a control diet from 1 to 4 months of age, approximately 10 months before the appearance of striatal dopamine loss in this model. Unbiased weighted gene co-expression network analysis (WGCNA) of transcriptional changes revealed effects of cholesterol oximes on transcripts related to mitochondria, cytoprotection and anti-oxidant response in wild-type and transgenic mice, including increased transcription of stress defense (e.g. Prdx1, Prdx2, Glrx2, Hspa9, Pink1, Drp1, Trak1) and dopamine-related (Th, Ddc, Gch1, Dat, Vmat2, Drd2, Chnr6a) genes. Even at this young age transgenic mice showed alterations in transcripts implicated in mitochondrial function and oxidative stress (e.g. Bcl-2, Bax, Casp3, Nos2), and both drugs normalized about 20% of these alterations. Young Thy1-aSyn mice exhibit motor deficits that differ from parkinsonism and are established before the onset of treatment; these deficits were not improved by cholesterol oximes. However, high doses of TRO40303 improved olfaction and produced the same effects as dopamine agonists on a challenging beam test, specifically an increase in footslips, an observation congruent with its effects on transcripts involved in dopamine synthesis. High doses of TRO19622 increased alpha-synuclein aggregates in the substantia nigra; this effect, not seen with TRO40303 was inconsistent and may represent a protective mechanism as in other neurodegenerative diseases. Overall, the results suggest that cholesterol oximes, while not improving early effects of alpha-synuclein overexpression on motor behavior or pathology, may ameliorate the function and resilience of dopaminergic neurons in vivo and support further studies of neuroprotection in models with dopaminergic cell loss.

Introduction

Parkinson's disease (PD) causes widespread pathology in brain but it is the progressive dysfunction and death of nigrostriatal dopaminergic neurons that is mainly responsible for the disabling motor deficits characteristic of the disease. Therefore, preserving the function and improving the survival of these dopaminergic neurons remain a therapeutic priority. Oxidative stress and mitochondrial dysfunction have been observed in patients with sporadic Parkinson's disease (PD) (Lin and Beal, 2006, Schapira and Jenner, 2011, Schapira et al., 1989). Mutations that cause rare familial forms of PD affect mitochondria clearance or function (Subramaniam and Chesselet, 2013), and alpha-synuclein, a protein related to both familial and sporadic forms of PD (Hardy, 2010, Pankratz et al., 2009), affects mitochondrial function (Devi et al., 2008, Loeb et al., 2010, Nakamura, 2013). Despite the likely involvement of mitochondria in the disease, previous use of anti-oxidants in PD has been disappointing (Stocchi and Olanow, 2013), however compounds that directly target mitochondria have not been extensively examined.

Cholesterol oximes, a novel family of neuroprotective compounds, were originally identified in a cell-based assay of neurotrophic deprivation in primary motor neurons (Bordet et al., 2007). The lead compound, olesoxime (TRO19622; cholest-4-en-3-one, oxime), provides similar protection to human iPSC-derived motor neurons (Yang et al., 2013) and is active in multiple in vivo models of neurodegeneration (Bordet et al., 2007, Bordet et al., 2008, Magalon et al., 2012, Martin et al., 2011, Sunyach et al., 2012, Xiao et al., 2009, Xiao et al., 2012). TRO40303 (3,5-seco-4-nor-cholestan-5-one oxime-3-ol) was derived from a chemistry optimization program based on the structure of TRO19622 and has pronounced cytoprotective effects and preserves cardiac tissue in a rat model of cardiac ischemia–reperfusion injury (Schaller et al., 2010). These cholesterol oximes access brain after oral administration, are safe, and have moved forward into clinical trials (Le Lamer et al., 2014, Lenglet et al., 2014). They bind to mitochondrial outer membrane proteins, the 18 kDa translocator protein (TSPO) and the voltage-dependent anion channel (VDAC, TRO19622 only), and reduce oxidative stress-induced mitochondrial permeability transition (mPTP), and the release of pro-apoptotic factors (Bordet et al., 2007, Schaller et al., 2010). Previous studies with TRO compounds have provided evidence for stabilization of mitochondrial function under different disease conditions (Bordet et al., 2007, de Tassigny et al., 2013, Gouarne et al., 2013).

The goals of the present study were 1): to examine the molecular effects of cholesterol oximes on dopaminergic neurons; and 2) test their ability to reverse alpha-synuclein induced gene alterations in these neurons. We have used a well-characterized mouse model of alpha-synuclein over-expression in which full-length, human, wild-type alpha-synuclein is expressed under the Thy-1 promoter (Thy1-aSyn mice; Chesselet et al., 2012, Rockenstein et al., 2002). The Thy1-aSyn mice show a loss of striatal dopamine by 14 months, providing a long window of time to assess the molecular mechanisms that may lead to nigrostriatal pathology. We used young adult wildtype mice and Thy1-aSyn littermates as a cost-effective initial approach to study the effects of chronic administration of cholesterol oximes on dopaminergic neurons under physiological versus disease condition. TRO19622 or TRO40303 or a control diet was administered in food pellets to wildtype mice or to Thy1-aSyn mice from 1 to 3 months of age. We assessed the behavioral and pathological effects of alpha-synuclein over-expression observed at this early age in Thy1-aSyn mice (Chesselet et al., 2012), followed by an analysis of transcriptional changes in laser captured nigrostriatal dopaminergic neurons. At a dose that reached expected pharmacologically active levels in brain based on in vitro studies, TRO40303 had a marked effect on transcripts involved in neuroprotection and dopamine synthesis in wild-type mice, and to a lesser extent, in Thy1-aSyn mice. Although they did not improve early behavioral and pathological effects of alpha-synuclein over-expression, both compounds normalized a significant fraction of transcript changes in transgenic mice, with TRO40303 having the stronger effect.