Final Report Summary - DOPAMINE NEURON CODE (Developmental Transcription Factors in Dopamine Neuron Maintenance)
A fundamental problem in biology has been to understand how the tremendous cellular diversity within the central nervous system is generated during embryonic development. One cell type that has received considerable attention in these studies are midbrain dopamine (mDA) neurons since they are important for physiological functions such as motor control, cognition and reward, and since their degeneration is a hallmark of Parkinson's disease (PD). Transcription factors important for neuronal specification and differentiation often remain expressed in mature neurons but it remains unknown whether they are also important for the maintenance of neurons throughout life.
We first generated expression data for a list of candidate transcription factors to be expressed in late maturation and adult mDA neurons. We next focused on Lmx1a and Lmx1b, two key transcription factors required for the early specification of mDA neurons that remain expressed in mature mDA neurons and for which gene polymorphisms have been associated with PD. To directly establish how Lmx1a/Lmx1b function in postmitotic DA neurons and if dysfunction of these transcription factors may contribute to disease, we generated specific midbrain-DA-neuron conditional knockout mice (cKO). Lmx1a/b postmitotic ablation results in a significant loss of midbrain DA neurons and DA nerve terminals in the striatum, accompanied by depletion of DA and DA-metabolites levels in the striatum, nucleus accumbens (NAcc), prefrontal cortex (PFC) and hippocampus. These PD-like features correlate with progressive impairments in motor behaviour and olfaction. In addition, short-term memory formation and hippocampal synaptic plasticity are altered, further supporting a severe dysfunction of mDA cells. Electron microscopy studies show abnormally enlarged presynaptic DA terminals filled with vesicles and a severe reduction in the number of lipofucsin granules present in substantia nigra mDA neurons. Both ultrastructural alterations suggest a dysfunction of the autophagic-lysosomal pathway (ALP) in Lmx1a/b ablated mice, similar to what has been observed in the brains of PD patients and in several animal models of PD.
To distinguish Lmx1a/Lmx1b functions in early postmitotic and mature DA neurons, we generated tamoxifen inducible specific midbrain-DA-neuron conditional knockout mice. Lmx1a/b ablation at 4 weeks of age results in no major cell loss nor striatal DA terminal loss up to 18 months after ablation, and behavioural characterisation reveals only a dysfunction in olfaction. Importantly, however, HPLC measurements show a severe reduction in DA levels in the striatum, NAcc and PFC; and electron microscopy studies show abnormal presynaptic DA terminals in tamoxifen inducible Lmx1a/b cKO. Our results point to an early dysfunction of ALP in Lmx1a/b ablated mice that disturbs synaptic function and compromises the functionality of the dopamine system. Analysis of human post-mortem substantia nigra cells shows decreased LMX1B expression in a subset of PD patients relative to controls, suggesting that the pathologic mechanism identified in Lmx1a/b cKO might be relevant to PD pathogenesis.
Finally, we aimed at understanding which are the molecular events downstream Lmx1a/b. We first did acute lentiviral-mediated down-regulation of Lmx1b in mouse embryonic ventral midbrain primary cultures followed by quantitiative PCR, and identified a set of genes involved in ALP that are directly regulated by Lmx1b. One of such genes is the master ALP transcription factor TFEB, whose promoter has several putative Lmx1-binding sites. Second, we performed global RNA-sequencing from FACS-sorted mDA neurons derived from a Pitx3-eGFP mouse embryonic stem cell line after Lmx1b downregulation. The analysis of this data set will allow us to identify all gene expression changes that occur in a maturing mDA neuron upon loss of Lmx1b.
Taken together, our findings reveal a sustained requirement of developmental transcription factors for the maintenance of midbrain DA neurons, uncover an unexpected role for Lmx1b in the transcriptional regulation of the autophagic-lysosomal pathway, and suggest that adult Lmx1b dysfunction is associated with early features of PD pathogenesis.
We believe our results have a broad socio-economic impact. First, because they improve our understanding of the pathologic events that contribute to Parkinson's disease onset and progression. Second because the identification of an autophagic-lysosomal dysfunction as an early pre-symptomatic event has potential diagnostic power and could be of great clinical benefit. And third, the identification of Lmx1b as a critical transcriptional regulator whose dysfunction compromises the function of the dopamine system points to this transcription factor pathway as a potential new drug target.
We first generated expression data for a list of candidate transcription factors to be expressed in late maturation and adult mDA neurons. We next focused on Lmx1a and Lmx1b, two key transcription factors required for the early specification of mDA neurons that remain expressed in mature mDA neurons and for which gene polymorphisms have been associated with PD. To directly establish how Lmx1a/Lmx1b function in postmitotic DA neurons and if dysfunction of these transcription factors may contribute to disease, we generated specific midbrain-DA-neuron conditional knockout mice (cKO). Lmx1a/b postmitotic ablation results in a significant loss of midbrain DA neurons and DA nerve terminals in the striatum, accompanied by depletion of DA and DA-metabolites levels in the striatum, nucleus accumbens (NAcc), prefrontal cortex (PFC) and hippocampus. These PD-like features correlate with progressive impairments in motor behaviour and olfaction. In addition, short-term memory formation and hippocampal synaptic plasticity are altered, further supporting a severe dysfunction of mDA cells. Electron microscopy studies show abnormally enlarged presynaptic DA terminals filled with vesicles and a severe reduction in the number of lipofucsin granules present in substantia nigra mDA neurons. Both ultrastructural alterations suggest a dysfunction of the autophagic-lysosomal pathway (ALP) in Lmx1a/b ablated mice, similar to what has been observed in the brains of PD patients and in several animal models of PD.
To distinguish Lmx1a/Lmx1b functions in early postmitotic and mature DA neurons, we generated tamoxifen inducible specific midbrain-DA-neuron conditional knockout mice. Lmx1a/b ablation at 4 weeks of age results in no major cell loss nor striatal DA terminal loss up to 18 months after ablation, and behavioural characterisation reveals only a dysfunction in olfaction. Importantly, however, HPLC measurements show a severe reduction in DA levels in the striatum, NAcc and PFC; and electron microscopy studies show abnormal presynaptic DA terminals in tamoxifen inducible Lmx1a/b cKO. Our results point to an early dysfunction of ALP in Lmx1a/b ablated mice that disturbs synaptic function and compromises the functionality of the dopamine system. Analysis of human post-mortem substantia nigra cells shows decreased LMX1B expression in a subset of PD patients relative to controls, suggesting that the pathologic mechanism identified in Lmx1a/b cKO might be relevant to PD pathogenesis.
Finally, we aimed at understanding which are the molecular events downstream Lmx1a/b. We first did acute lentiviral-mediated down-regulation of Lmx1b in mouse embryonic ventral midbrain primary cultures followed by quantitiative PCR, and identified a set of genes involved in ALP that are directly regulated by Lmx1b. One of such genes is the master ALP transcription factor TFEB, whose promoter has several putative Lmx1-binding sites. Second, we performed global RNA-sequencing from FACS-sorted mDA neurons derived from a Pitx3-eGFP mouse embryonic stem cell line after Lmx1b downregulation. The analysis of this data set will allow us to identify all gene expression changes that occur in a maturing mDA neuron upon loss of Lmx1b.
Taken together, our findings reveal a sustained requirement of developmental transcription factors for the maintenance of midbrain DA neurons, uncover an unexpected role for Lmx1b in the transcriptional regulation of the autophagic-lysosomal pathway, and suggest that adult Lmx1b dysfunction is associated with early features of PD pathogenesis.
We believe our results have a broad socio-economic impact. First, because they improve our understanding of the pathologic events that contribute to Parkinson's disease onset and progression. Second because the identification of an autophagic-lysosomal dysfunction as an early pre-symptomatic event has potential diagnostic power and could be of great clinical benefit. And third, the identification of Lmx1b as a critical transcriptional regulator whose dysfunction compromises the function of the dopamine system points to this transcription factor pathway as a potential new drug target.