Silencing Parkinson's risk allele Rit2 sex-specifically compromises motor function and dopamine neuron viability.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease and arises from dopamine (DA) neuron death selectively in the substantia nigra pars compacta (SNc). Rit2 is a reported PD risk allele, and recent single cell transcriptomic studies identified a major RIT2 cluster in PD DA neurons, potentially linking Rit2 expression loss to a PD patient cohort. However, it is still unknown whether Rit2 loss itself impacts DA neuron function and/or viability. Here we report that conditional Rit2 silencing in mouse DA neurons drove motor dysfunction that occurred earlier in males than females and was rescued at early stages by either inhibiting the DA transporter (DAT) or with L-DOPA treatment. Motor dysfunction was accompanied by decreased DA release, striatal DA content, phenotypic DAergic markers, DA neurons, and DAergic terminals, with increased pSer129-alpha synuclein and pSer935-LRRK2 expression. These results provide clear evidence that Rit2 loss is causal for SNc cell death and motor dysfunction, and reveal key sex-specific differences in the response to Rit2 loss.

Rit2 silencing in dopamine neurons drives a progressive Parkinsonian phenotype.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease and arises from dopamine (DA) neuron death selectively in the substantia nigra pars compacta (SNc). Rit2 is a reported PD risk allele, and recent single cell transcriptomic studies identified a major RIT2 cluster in PD DA neurons, potentially linking Rit2 expression anomalies to a PD patient cohort. However, it is still unknown whether Rit2 loss itself is causative for PD or PD-like symptoms. Here we report that conditional Rit2 silencing in mouse DA neurons drove a progressive motor dysfunction that was more rapid in males than females and was rescued at early stages by either inhibiting the DA transporter (DAT) or with L-DOPA treatment. Motor dysfunction was accompanied by decreases in DA release, striatal DA content, phenotypic DAergic markers, and a loss of DA neurons, with increased pSer129-alpha synuclein expression. These results provide the first evidence that Rit2 loss is causal for SNc cell death and a PD-like phenotype, and reveal key sex-specific differences in the response to Rit2 loss.

Rit2 silencing in dopamine neurons drives a Parkinsonian phenotype.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease and arises from dopamine (DA) neuron death selectively in the substantia nigra pars compacta (SNc). Rit2 is a reported PD risk allele, and recent single cell transcriptomic studies identified a major RIT2 cluster in PD DA neurons, potentially linking Rit2 expression loss to a PD patient cohort. However, it is still unknown whether Rit2 loss itself is causative for PD or PD-like symptoms. Here we report that conditional Rit2 silencing in mouse DA neurons drove motor dysfunction that occurred earlier in males than females and was rescued at early stages by either inhibiting the DA transporter (DAT) or with L-DOPA treatment. Motor dysfunction was accompanied by decreased DA release, striatal DA content, phenotypic DAergic markers, DA neurons, and DAergic terminals, with increased pSer129-alpha synuclein and pSer935-LRRK2 expression. These results provide the first evidence that Rit2 loss is causal for SNc cell death and a PD-like phenotype, and reveal key sex-specific differences in the response to Rit2 loss.

Presynaptic Gq-coupled receptors drive biphasic dopamine transporter trafficking that modulates dopamine clearance and motor function.

Extracellular dopamine (DA) levels are constrained by the presynaptic DA transporter (DAT), a major psychostimulant target. Despite its necessity for DA neurotransmission, DAT regulation in situ is poorly understood, and it is unknown whether regulated DAT trafficking impacts dopaminergic signaling and/or behaviors. Leveraging chemogenetics and conditional gene silencing, we found that activating presynaptic Gq-coupled receptors, either hM3Dq or mGlu5, drove rapid biphasic DAT membrane trafficking in ex vivo striatal slices, with region-specific differences between ventral and dorsal striata. DAT insertion required D2 DA autoreceptors and intact retromer, whereas DAT retrieval required PKC activation and Rit2. Ex vivo voltammetric studies revealed that DAT trafficking impacts DA clearance. Furthermore, dopaminergic mGlu5 silencing elevated DAT surface expression and abolished motor learning, which was rescued by inhibiting DAT with a subthreshold CE-158 dose. We discovered that presynaptic DAT trafficking is complex, multimodal, and region specific, and for the first time, we identified cell autonomous mechanisms that govern presynaptic DAT tone. Importantly, the findings are consistent with a role for regulated DAT trafficking in DA clearance and motor function.

Histone deacetylase inhibitors modulate miRNA and mRNA expression, block metaphase, and induce apoptosis in inflammatory breast cancer cells.

To develop new therapies for inflammatory breast cancer (IBC) we have compared the effects of two hydroxamic acid-based histone deacetylase (HDAC) inhibitors, CG-1521 and Trichostatin A (TSA) on the biology of two IBC cell lines: SUM149PT and SUM190PT. CG-1521 and TSA induce dose (0-10 µM) and time-dependent (0-96 h) increases in the proportion of cells undergoing cell cycle arrest and apoptosis in the presence or absence of 17ß-estradiol. In SUM 149PT cells, both CG-1521 and TSA increase the levels of acetylated α-tubulin; however the morphological effects are different: CG-1521 blocks mitotic spindle formation and prevents abscission during cytokinesis while TSA results in an increase in cell size. In SUM190PT cells CG-1521 does not cause an increase in acetylated-α-tubulin and even though TSA significantly increases the levels of acetylated tubulin, neither inhibitor alters the morphology of the cells. Microarray analysis demonstrates that CG-1521 modulates the expression of 876 mRNAs and 63 miRNAs in SUM149PT cells, and 1227 mRNAs and 35 miRNAs in SUM190PT cells. Only 9% of the genes are commonly modulated in both cell lines, suggesting that CG-1521 and TSA target different biological processes in the two cell lines most likely though the inhibition of different HDACs in these cell lines. Gene ontology (GO) analysis reveals that CG-1521 affects the expression of mRNAs that encode proteins associated with the spindle assembly checkpoint, chromosome segregation, and microtubule-based processes in both cell lines and has cell-type specific effects on lipid biosynthesis, response to DNA damage, and cell death.