This chapter reviews vertebrate α-synuclein transgenic mouse models which have led to substantial advances in understanding the role of α-synuclein in pathogenic processes that are relevant to ...Parkinson's disease (PD). Mutations in the gene coding for leucine rich repeat kinase-2 (LRRK2) also lead to autosomal-dominant PD. LRRK2 is a large protein containing multiple functional domains including a GTPase and kinase domain. Of the various α-synuclein transgenic mice that have been generated over the years, the models using the murine Thy-1 promoter and the murine prion promoter recapitulate most if not all the pathogenic features of PD, except for degeneration of dopamine neurons. Transgenic mice overexpressing α-synuclein under either promoter develop α-synucleinopathy in neurons and overlapping brain regions with predominate pathology in spinal motor neurons, deep cerebellar nuclei, pontine reticular nuclei and the red nucleus. The neuronal populations are particularly vulnerable to α-synuclein-induced neurodegeneration in mice. It is suggested that the α-synuclein transgenic models can be used to explore the molecular basis of cell death induced by α-synuclein.
Although the failure of the UPS has been implicated in both sporadic and familial forms of PD, it is unclear as to whether it is involved in the initiation or progression (or both) of the disease. It ...is hypothesized that mutations in parkin impair its normal ubiquitination activity, leading to an accumulation of proteins that overload the UPS and result in the selective death of neurons in the substantia nigra by a mechanism that is largely unknown. The paucity of Lewy bodies and therefore a lack of normal cellular defenses against excess levels of toxic proteins may manifest as early onset and severe neurodegeneration in parkin-associated PD. The pathogenic connection between the ubiquitination pathway and parkin-related PD has been established — the thrust should now be on understanding the mechanisms that cause and contribute to the development of disease.
Mitochondria and the nucleus play critical roles in cell death. The signaling between these two organelles is dynamic. Understanding the signaling process between the nucleus and the mitochondria in ...cell death programs could lead us to more options in the development of new therapeutic targets. We discussed in this chapter how AIF could be released from the mitochondria and how AIF could act in PARP-dependent cell death mechanism. The molecular mechanisms accounting for PARP-dependent cell death are still being investigated but mitochondrial release of AIF and its translocation to the nucleus appears to be essential in this death process. We speculate that the PARP-mediated depletion of NAD and ATP or novel signals from PARP over activation might trigger AIF translocation; however, the authentic factors and the legitimate mechanism of mitochondrial AIF release remain to be explored.
A unifying feature of neurodegenerative diseases is the abnormal accumulation and processing of mutant or damaged intra- and extracellular proteins; this leads to selective neuronal vulnerability and ...dysfunction. The ubiquitin-proteasomal pathway (UPP) is poised to play a central role in the processing of damaged and toxic proteins by ubiquitin-dependent proteolysis. The UPP can be overwhelmed in several neurodegenerative diseases. This results in the accumulation of toxic proteins and the formation of inclusions, and ultimately to neuronal dysfunction and cell death. Further analysis of the cellular and molecular mechanisms by which the UPP influences the detoxification of damaged and toxic proteins in neurodegenerative diseases could provide novel concepts and targets for the treatment and understanding of the pathogenesis of these devastating disorders.
The Stroke Preclinical Assessment Network (SPAN) is a multicenter preclinical trial platform using rodent models of transient focal cerebral ischemia to address translational failure in experimental ...stroke. In addition to centralized randomization and blinding and large samples, SPAN aimed to introduce heterogeneity to simulate the heterogeneity embodied in clinical trials for robust conclusions. Here, we report the heterogeneity introduced by allowing the 6 SPAN laboratories to vary most of the biological and experimental model variables and the impact of this heterogeneity on middle cerebral artery occlusion (MCAo) performance. We included the modified intention-to-treat population of the control mouse cohort of the first SPAN trial (n=421) and examined the biological and procedural independent variables and their covariance. We then determined their impact on the dependent variables cerebral blood flow drop during MCAo, time to achieve MCAo, and total anesthesia duration using multivariable analyses. We found heterogeneity in biological and procedural independent variables introduced mainly by the site. Consequently, all dependent variables also showed heterogeneity among the sites. Multivariable analyses with the site as a random effect variable revealed filament choice as an independent predictor of cerebral blood flow drop after MCAo. Comorbidity, sex, use of laser Doppler flow to monitor cerebral blood flow, days after trial onset, and maintaining anesthesia throughout the MCAo emerged as independent predictors of time to MCAo. Total anesthesia duration was predicted by most independent variables. We present with high granularity the heterogeneity introduced by the biological and model selections by the testing sites in the first trial of cerebroprotection in rodent transient filament MCAo by SPAN. Rather than trying to homogenize all variables across all sites, we embraced the heterogeneity to better approximate clinical trials. Awareness of the heterogeneity, its sources, and how it impacts the study performance may further improve the study design and statistical modeling for future multicenter preclinical trials.
One approach to studying the functional role of individual NMDA receptor subunits involves the reduction in the abundance of the protein subunit in neurons. We have pursued a strategy to achieve this ...goal that involves the use of a small guide RNA which can lead to the destruction of the mRNA for a specific receptor subunit. We designed a small RNA molecule, termed ‘external guide sequence’ (EGS), which binds to the NR1 mRNA and directs the endonuclease RNase P to cleave the target message. This EGS has exquisite specificity and directed the RNase P‐dependent cleavage at the targeted location within the NR1 mRNA. To improve the efficiency of this EGS, an in vitro evolution strategy was employed which led to a second generation EGS that was 10 times more potent than the parent molecule. We constructed an expression cassette by flanking the EGS with self‐cleaving ribozymes and this permitted generation of the specified EGS RNA sequence from any promoter. Using a recombinant Herpes simplex virus (HSV), we expressed the EGS in neurons and showed the potency of the EGS to reduce NR1 protein within neurons. In an excitotoxicity assay, we showed that expression of the EGS in cortical neurons is neuroprotective. Our results demonstrate the utility of EGSs to reduce the expression of any gene (and potentially any splice variant) in neurons.
