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.

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 4months of age, approximately 10months 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.

Cognitive deficits in a mouse model of pre-manifest Parkinson's disease.

Early cognitive deficits are increasingly recognized in patients with Parkinson's disease (PD), and represent an unmet need for the treatment of PD. These early deficits have been difficult to model in mice, and their mechanisms are poorly understood. α-Synuclein is linked to both familial and sporadic forms of PD, and is believed to accumulate in brains of patients with PD before cell loss. Mice expressing human wild-type α-synuclein under the Thy1 promoter (Thy1-aSyn mice) exhibit broad overexpression of α-synuclein throughout the brain and dynamic alterations in dopamine release several months before striatal dopamine loss. We now show that these mice exhibit deficits in cholinergic systems involved in cognition, and cognitive deficits in domains affected in early PD. Together with an increase in extracellular dopamine and a decrease in cortical acetylcholine at 4-6 months of age, Thy1-aSyn mice made fewer spontaneous alternations in the Y-maze and showed deficits in tests of novel object recognition (NOR), object-place recognition, and operant reversal learning, as compared with age-matched wild-type littermates. These data indicate that cognitive impairments that resemble early PD manifestations are reproduced by α-synuclein overexpression in a murine genetic model of PD. With high power to detect drug effects, these anomalies provide a novel platform for testing improved treatments for these pervasive cognitive deficits.

A Molecular Tweezer Ameliorates Motor Deficits in Mice Overexpressing α-Synuclein.

Aberrant accumulation and self-assembly of α-synuclein are tightly linked to several neurodegenerative diseases called synucleinopathies, including idiopathic Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Deposition of fibrillar α-synuclein as insoluble inclusions in affected brain cells is a pathological hallmark of synucleinopathies. However, water-soluble α-synuclein oligomers may be the actual culprits causing neuronal dysfunction and degeneration in synucleinopathies. Accordingly, therapeutic approaches targeting the toxic α-synuclein assemblies are attractive for these incurable disorders. The "molecular tweezer" CLR01 selectively remodels abnormal protein self-assembly through reversible binding to Lys residues. Here, we treated young male mice overexpressing human wild-type α-synuclein under control of the Thy-1 promoter (Thy1-aSyn mice) with CLR01 and examined motor behavior and α-synuclein in the brain. Intracerebroventricular administration of CLR01 for 28 days to the mice improved motor dysfunction in the challenging beam test and caused a significant decrease of buffer-soluble α-synuclein in the striatum. Proteinase-K-resistant, insoluble α-synuclein deposits remained unchanged in the substantia nigra, whereas levels of diffuse cytoplasmic α-synuclein in dopaminergic neurons increased in mice receiving CLR01 compared with vehicle. More moderate improvement of motor deficits was also achieved by subcutaneous administration of CLR01, in 2/5 trials of the challenging beam test and in the pole test, which requires balance and coordination. The data support further development of molecular tweezers as therapeutic agents for synucleinopathies.

Effects of decreased dopamine transporter levels on nigrostriatal neurons and paraquat/maneb toxicity in mice.

How genetic variations in the dopamine transporter (DAT) combined with exposure to environmental toxins modulate the risk of Parkinson's disease remains unclear. Using unbiased stereology in DAT knock-down mice (DAT-KD) and wild-type (WT) littermates, we found that decreased DAT caused a loss of tyrosine hydroxylase-positive (dopaminergic) neurons in subregions of the substantia nigra pars compacta at 3-4 days, 5 weeks, and 18 months of age. Both genotypes lost dopaminergic neurons with age and remaining neurons at 11 months were resilient to paraquat/maneb. In 5-week-old mice, the toxins decreased substantia nigra pars compacta dopaminergic neurons in both genotypes but less in DAT-KD. Regional analysis revealed striking differences in the subsets of neurons affected by low DAT, paraquat/maneb, and aging. In particular, we show that a potentially protective effect of low DAT against toxin exposure is not sufficient to reduce death of all nigrostriatal dopaminergic neurons. Thus, different regional vulnerability of nigrostriatal dopaminergic neurons may contribute to an increased risk of developing Parkinson's disease when multiple factors are combined.

A GCase chaperone improves motor function in a mouse model of synucleinopathy.

Mutation of the lysosomal hydrolase acid-ß-glucosidase (GCase), which leads to reduced GCase activity, is one of the most frequent genetic risk factors for Parkinson's disease (PD) and promotes α-synuclein accumulation in the brain, a hallmark of PD and other synucleinopathies. Whether targeting GCase pharmacologically is a valid therapeutic strategy for sporadic PD in the absence of GCase mutation is unknown. We have investigated whether increasing the stability, trafficking, and activity of wild-type GCase could be beneficial in synucleinopathies by administering the pharmacological chaperone AT2101 (afegostat-tartrate, isofagomine) to mice that overexpress human wild-type α-synuclein (Thy1-aSyn mice). AT2101 administered orally for 4 months to Thy1-aSyn mice improved motor and nonmotor function, abolished microglial inflammatory response in the substantia nigra, reduced α-synuclein immunoreactivity in nigral dopaminergic neurons, and reduced the number of small α-synuclein aggregates, while increasing the number of large α-synuclein aggregates. These data support the further investigation of pharmacological chaperones that target GCase as a therapeutic approach for sporadic PD and other synucleinopathies, even in the absence of glucocerebrosidase mutations.