Funded Studies
Objective/Rationale:
Multiple evidence supports the notion that a malfunction of the ubiquitin-proteasome system and autophagy plays a crucial role in protein misfolding and aggregation in Parkinson's disease (PD). One important player in protein ubiquitination is Skp1, an ubiquitin-proteasome/E3-ligase component. The main goal of this project is to determine whether SKP1A constitutes a new molecular target to prevent/ameliorate dopamine (DA) neuronal damage in relevant preclinical models of PD.
Project Description:
To clarify whether Skp1 plays a role in DA neuronal function two approaches will be applied: The first will assess whether the reduction of Skp1 in the pre-clinical model substantia nigra using lentiviruses will cause age-dependent loss of nigral dopaminergic cells, protein aggregates formation and impairment of the integrity of the nigrostriatal axis with emergence of motor abnormalities. In the second approach SKP1A gene expression will be increased in both toxin and genetic models of PD by nigral infection with viral vectors expressing SKP1A and subsequent damage of pre-clinical models with MPTP, or by simultaneous infection of pre-clinical models with viral vectors encoding SKP1A and human alpha-synuclein. It is expected that overexpression of Skp1 will decrease abnormal synuclein-containing cytoplasmic inclusion, neuritic pathology and motor disability.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
Our studies have demonstrated a selective reduction in the levels of Skp1 gene and protein in the substantia nigra pars compacta of sporadic cases of PD. The identification of parkinsonism-causing recessive mutations in the Skp1-interacting protein PARK15/Fbxo7, suggests a role for Skp1 in both sporadic and familial cases of PD. Thus, approaches targeted at enhancing Skp1 function (of the gene therapy or a pharmacological agent), could be relevant to treat or slow progression of PD.
Anticipated Outcome:
Results from this study will help to elucidate the beneficial role of Skp1 in DA neuron survival and to validate Skp1 as a novel therapeutic target in PD. If it would be possible to replicate the adult onset, progressive neurodegeneration in the nigro-striatal axis and motor deficits, the Skp1-deficient pre-clinical model may serve as a model of sporadic PD providing a reliable "platform" to develop new therapeutic interventions.
Final Outcome
The main goal of this project was to determine whether Skp1 constitutes a new molecular target to prevent/ameliorate dopamine (DA) neuronal damage in relevant preclinical models Parkinson's disease (PD). To genetically manipulate the brain levels of Skp1, pre-clinical models underwent stereotactic surgical procedure using special viral vectors to decrease SKP1A gene expression specifically in the SN. The pre-clinical models were tracked for the emergence of motor abnormalities reminiscent of human PD, such as muscle strength, motor coordination and balance up to a period of 12 months.
The results show that targeted reduction of SKP1A in the SN did not significantly alter motor function in the course of 3-12 month post-operation time period when compared to those injected with a non-SKP1A carrying virus. These findings indicate that the that the sole reduction in Skp1 is not sufficient to engender essential features of PD pathology, even when reduced at middle age stage, suggesting that age does not modify phenotype characteristics that might have been caused by a deficiency of Skp1.
We have considered the possibility that the pre-clinical model may not be the most appropriate model to emulate motor deficits induced by a genetic reduction of SKP1A in the SN. To test this, we have selected the fly Drosophila Melanogaster as an alternative model to reduce brain levels of SKPA, the ortholog of SKP1A, in either the whole neuronal population, or specifically in the TH expressing neurons. Our results revealed that the knockdown of SKPA caused a marked shortening in the lifespan of the flies and a progressive dysfunction in their climbing motor ability, with gradual loss of DA-producing cells. This effect was evident either when SKPA was deficient from early developmental stage or when its levels were reduced at adultness. Thus, genetic manipulation in Drosophila may provide a valid model to shed light on Skp1 role in neuroprotection, protein aggregation and other mechanistic features. In conclusion, the potential of SKP1 as a genetic modifier in PD cannot be ruled out and more experimental data, especially in other pre-clinical models, is needed before a clearer picture is obtained.