Dopaminergic control of autophagic-lysosomal function implicates Lmx1b in Parkinson's disease

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mDA neurons constitute the main dopaminergic cell population in the CNS1. Degeneration of these cells in the substantia nigra pars compacta is a characteristic feature of Parkinson’s disease (PD), one of the most common neurological disorders. PD is characterized by the appearance of α-synuclein-containing Lewy bodies in mDA neurons and other neurons of the brain and by a progressive pathology that ultimately leads to neuron death. Early pathology, such as reduced stri-atal dopamine (DA), diminished expression of several mDA neuron– specific proteins and abnormal accumulation of α-synuclein and other proteins, is believed to occur long before neurons actually die2. Thus, understanding disrupted protein-degradation pathways and maintenance of mDA neuron–specific properties at early stages of disease progression will be essential in elucidating critical cell pathological events.

Developmental transcription factors that are responsible for the acquisition of differentiated neuron characteristics are in many cases also expressed in adult neurons, raising questions as to how they con-tribute to the active maintenance of neuronal identities3,4. Notably, ablation of the transcription factor gene encoding Nurr1 in adult mDA neurons leads to a phenotype that shows striking resemblance to early features of PD, including reduced striatal DA, degeneration of target innervation and diminished expression of nuclearly encoded mitochondrial genes5. The transcription factors Foxa1 and Foxa2 are important for maintained Nurr1 (Nr4a2) expression in mDA neu-rons, and conditional ablation of these two factors in maturing mDA neurons results in abnormalities that are similar to those observed in Nurr1 conditional knockout mice6. In addition, the transcription

factor Otx2 has been shown to be important for the maintenance of mDA neuron subtype-specific characteristics in the ventral teg-mental area (VTA)7. The function of developmental transcription factors may thus be linked to PD, a conclusion that is supported by genome-wide association studies indicating that genetic variants of human transcription factors, including LMX1A and LMX1B, may contribute to PD8–13.

Lmx1a and Lmx1b are two highly related LIM homeodomain tran-scription factors that are essential in developing mDA neurons14,15. Lmx1a and Lmx1b are first expressed in ventral midbrain proliferat-ing neural progenitor cells, where they specify uncommitted neural stem cells into cells that eventually differentiate into mDA neurons16. Combined null mutations in both, but not in the individual, genes result in disrupted Wnt1 signaling, decreased progenitor cell prolif-eration and decreased neurogenesis, leading to essentially abolished generation of mDA neurons14,15. Their central role in cell specifica-tion has also been demonstrated by the robust generation of mDA neurons in vitro after forced expression of Lmx1a in both mouse and human pluripotent stem cells, results that are of potential relevance in cell replacement in PD16–19. In normal development, Lmx1a and Lmx1b continue to be expressed in postmitotic differentiating mDA neurons20–22. However, their function after specifying dividing neural progenitors remains unknown.

Here we assessed the function of Lmx1a and Lmx1b (referred to as Lmx1a/b) in maturing and adult mDA neurons by analyzing the consequences of mDA neuron–specific ablation in condi-tional knockout mice. Our findings link the function of Lmx1b to

1Ludwig Institute for Cancer Research, Stockholm, Sweden. 2Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden. 3Neurodegenerative Diseases Group, Vall d’Hebron Research Institute-CIBERNED, Barcelona, Spain. 4Department of Clinical Neuroscience, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden. 5Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden. 6Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden. 7These authors contributed equally to this work. Correspondence should be addressed to A.L. ([email protected]) or T.P. ([email protected]).

Received 30 January; accepted 19 March; published online 27 April 2015; doi:10.1038/nn.4004

Dopaminergic control of autophagic-lysosomal function implicates Lmx1b in Parkinson’s diseaseAriadna Laguna1–3, Nicoletta Schintu4,7, André Nobre1,7, Alexandra Alvarsson4, Nikolaos Volakakis1, Jesper Kjaer Jacobsen1, Marta Gómez-Galán5, Elena Sopova5, Eliza Joodmardi1, Takashi Yoshitake6, Qiaolin Deng2, Jan Kehr6, Johan Ericson2, Per Svenningsson4, Oleg Shupliakov5 & Thomas Perlmann1,2

The role of developmental transcription factors in maintenance of neuronal properties and in disease remains poorly understood. Lmx1a and Lmx1b are key transcription factors required for the early specification of ventral midbrain dopamine (mDA) neurons. Here we show that conditional ablation of Lmx1a and Lmx1b after mDA neuron specification resulted in abnormalities that show striking resemblance to early cellular abnormalities seen in Parkinson’s disease. We found that Lmx1b was required for the normal execution of the autophagic-lysosomal pathway and for the integrity of dopaminergic nerve terminals and long-term mDA neuronal survival. Notably, human LMX1B expression was decreased in mDA neurons in brain tissue affected by Parkinson’s disease. Thus, these results reveal a sustained and essential requirement of Lmx1b for the function of midbrain mDA neurons and suggest that its dysfunction is associated with Parkinson’s disease pathogenesis.

http://www.nature.com/doifinder/10.1038/nn.4004

http://www.nature.com/natureneuroscience/

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mechanisms that are central for cellular homeostasis and that are tightly linked to the onset of PD pathology.

RESULTSLmx1a and Lmx1b expression in postmitotic mDA neuronsWe first investigated how Lmx1a/b are expressed in postmitotic neu-rons in both rodents and humans. Coronal mouse midbrain sections from mice ranging from embryonic day (E) 15.5 to 20 months old were analyzed by in situ hybridization. An overlapping expression of both genes coinciding with tyrosine hydroxylase (Th) mRNA expression was evident (Fig. 1a), but the temporal dynamics of expres-sion differed between the two genes. While Lmx1b continued to be strongly expressed at all stages, Lmx1a expression decreased rapidly at postnatal stages and was barely detectable at 6 months and undetec-table at 20 months. We confirmed the expression pattern by real-time quantitative PCR (RT-qPCR) from dissected ventral midbrain tissue (Fig. 1b). Thus, a high level of expression of Lmx1b, but not Lmx1a, is retained in adult neurons in mice.

LMX1B was also expressed in human postmitotic neuromelanin-containing mDA neurons (Fig. 1c). Consistent with results in mouse,

expression of LMX1A was not detected (Supplementary Fig. 1). To investigate whether the expression level of LMX1B may be altered in PD, we analyzed expression in postmortem PD brain samples and compared them to age-matched controls (Fig. 1c and Supplementary Table 1). In line with the possibility that deregulated transcrip-tion factor function may contribute to PD pathology, we observed decreased LMX1B expression in mDA neurons from postmortem PD brain samples (Fig. 1d). Thus, these observations in rodents and humans emphasize the importance of analyzing the roles of Lmx1a/b at stages following mDA neuron specification.

Behavioral and histological changes after loss of Lmx1a/bGiven the close structural relationship between Lmx1a and Lmx1b and the functional redundancy seen in mDA neuron specification14,15, we initially focused on the consequences of combined genetic ablation of Lmx1a and Lmx1b at stages following mDA neuron specification. Thus, we developed an animal model for Lmx1a/b conditional defi-ciency in postmitotic mDA neurons by crossing mouse strains that were double homozygous for loxP-flanked (‘floxed’) alleles of Lmx1a and Lmx1b with heterozygous mice expressing Cre recombinase

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Figure 1 Maintenance of Lmx1a and Lmx1b expression in postmitotic and adult DA neurons. (a) Representative nonradioactive in situ hybridization in ventral midbrain cryosections from C57BL6 mouse brains at the indicated time points (P0, newborn). Expression of tyrosine hydroxylase (Th) is shown as a reference to identify DA neurons. n = 2 animals per stage. Scale bar, 100 µm. (b) RT-qPCR analysis showing Lmx1a and Lmx1b mRNA expression in the ventral midbrain of C57BL6 embryos and mice at E15.5 and 3 and 18 months (m). Data are represented as mean ± s.e.m. of the fold change relative to the expression at E15.5 and normalized against Rpl19. n = 4 animals per stage. (c) Representative LMX1B immunostaining (blue; brown pigment is neuromelanin) in postmortem substantia nigra sections from three Parkinson’s disease (PD) patients and three age-matched healthy control subjects. Information and clinical data on PD patients and controls is included in Supplementary Table 1. Scale bar, 25 µm. (d) Densitometry quantification of LMX1B immunostaining intensity in neuromelanin-positive cells in postmortem substantia nigra sections from PD patients and control subjects (n = 20 to 65 neuromelanin-positive cells in each case; n = 3 control and n = 3 PD cases). Top, mean ± s.e.m. for the control and the PD group of cases. Bottom, individual intensity values for all cells analyzed (n = 124 control and n = 94 PD). Data are given in arbitrary units (AU). Mann-Whitney test ***P < 0.0005.

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under the regulatory control of the dopamine transporter gene (Slc6a3) locus (referred to as DAT-Cre)23. This generated animals in which both genes were either intact (cLmx1a/bCtrl) or ablated (cLmx1a/bDatCre) in mDA neurons. Slc6a3 is expressed in mDA neurons from approximately E13.5 (ref. 24). Consistently, immun-ofluorescence and qPCR analyses confirmed the efficiency of Lmx1a/b ablation in postmitotic mDA neurons in cLmx1a/bDatCre animals (Supplementary Fig. 1).

Next we analyzed adult (6-month-old) or aged (18-month-old) cLmx1a/bCtrl and cLmx1a/bDatCre animals in a battery of behavioral tests. A significant impairment in motor coordination was evident in adult Lmx1a/b-ablated mice, as determined by the pole test, and in aged Lmx1a/b-ablated mice, as determined by either beam traversal or pole test (Fig. 2a,b). An open field test showed a modest increase in locomotor activity in aged, but not in adult, cLmx1a/bDatCre animals, a behavior that has previously been seen after prenatal mDA neuron deficiency (see, for example, ref. 25) (Fig. 2c). Notably, non-motor behaviors as well, such as social olfaction measured by the wooden block test, were impaired in adult and aged cLmx1a/bDatCre animals (Fig. 2d). A cognitive test of novel object recognition showed memory impair-ment in adult cLmx1a/bDatCre relative to cLmx1a/bCtrl animals. Aged animals from both genotypes displayed a very poor performance in this test (Fig. 2e). No differences in performance were revealed in anxiety or depression-like tests (Supplementary Fig. 2). Thus, Lmx1a/b ablation in mDA neurons is associated with significant behavioral abnormalities related to dysfunctional DA neurotransmission.

Unbiased stereological cell counting of TH-positive and TH-negative Nissl-stained neurons in the ventral midbrain of young (2-month-old) and aged (18-month-old) mice indicated a significant and progressive loss of TH-positive neurons (Fig. 3a,b and Supplementary Fig. 3). Furthermore, ultrastructural analysis by electron microscopy demonstrated frequent examples of degenerating TH-positive neu-rons in young cLmx1a/bDatCre but not in controls (Fig. 3c; 11% ± 4% degenerating cells in cLmx1a/bDatCre mice versus 0% in cLmx1a/bCtrl mice, n = 70 cells in 2 animals per genotype).

We quantified TH- and DAT-immunopositive nerve terminals by densitometry in the mDA neuron target area in the dorsal and ventral

striatum. We noted a significant reduction in TH within both dorsal and ventral striatum in young and aged Lmx1a/b-ablated animals (Fig. 3d,e). DAT expression was also affected, but with a slower pro-gression, showing only modest reduction in young mice but a signifi-cant reduction in aged mice (Fig. 3e). The reduced striatal expression of TH and DAT appears to be the consequence of Lmx1b ablation, as we noted a similar reduction in these proteins from histological analysis performed on single cLmx1bDatCre, but not cLmx1aDatCre, animals (Supplementary Fig. 3).

We used high-pressure liquid chromatography (HPLC) of brain tissue extracts to determine the content of DA and the DA metabo-lites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in different brain areas. DA and its metabolites were signifi-cantly reduced in brain areas such as prefrontal cortex, hippocampus, dorsal striatum and nucleus accumbens in cLmx1a/bDatCre mice as compared to controls (Fig. 3f). These areas are innervated by mDA neuron axons originating in both the substantia nigra and the VTA, areas in which DA and metabolites were also reduced (Supplementary Fig. 3). DA and metabolites were not reduced in other areas such as the olfactory bulb and the cerebellum (Supplementary Fig. 3). Diminished striatal DA appears to be the consequence of perturbed Lmx1b function, as we observed reductions in single cLmx1bDatCre, but not cLmx1aDatCre, animals (Supplementary Fig. 3).

Since mDA neuron innervation was impaired, we speculated that modulation of synaptic transmission might also be affected in Lmx1a/b-ablated animals. Because VTA mDA neurons projecting to the hippocampus form a functional loop26 and because long-term potentiation (LTP) is a major model for assessing activity-depend-ent synaptic plasticity in the hippocampus27, we explored LTP in hippocampal slices from adult control and mutant mice. The data revealed normal basal synaptic transmission but an enhanced induced LTP of Shaffer collateral (SC)–CA1 pyramidal cell synapses in Lmx1a/b-ablated mice (Fig. 3g,h). Taken together, the analyses clearly indicate important Lmx1a/b functions in mDA neurons as reflected by behavioral, histological and functional abnormalities, including signs of neurodegeneration and abnormal synaptic responses in Lmx1a/b-ablated mice.

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Figure 2 Behavioral characterization of adult and old cLmx1a/bCtrl and cLmx1a/bDatCre mice. (a–e) Battery of behavioral tests performed with cLmx1a/bCtrl (cCtrl) and cLmx1a/bDatCre (cDatCre) mice. Beam traversal test (a; P = 0.0248) and pole test (b; T-turn adult, P = 0.0288; T-total adult, P = 0.0324; T-turn old, P = 0.0399; T-total old, P = 0.0493) indicate significant impairments of motor coordination and postural control. T-turn, time taken to orient downward at the top of the pole; T-total, total time to turn and descend the pole. Open field test (c) shows no major alteration in locomotor activity, though old cLmx1a/bDatCre mice show a slight hyperactivity (P = 0.0473), measured as the total distance traveled in the arena. Wooden block test (d; adult, P = 0.0392; old, P = 0.0001) reveals impaired social olfactory acuity. The investigatory index was calculated as the percentage of time spent with the wooden block scented with its own bedding minus the percentage of time spent with a block scented with bedding from another cage during a 2-min session. Novel object recognition test (e; P = 0.0474) reveals impaired short-term memory formation. The discrimination index was calculated as the cumulative time spent exploring both objects during the test session minus the difference between the exploratory times of the novel object (N) and the previously presented familiar object (F) respectively: (TimeN + TimeF) − (TimeN − TimeF). Adult mice were age 6–9 months and old mice 18–21 months. n = 8 or 9 cCtrl and 13 or 14 cDatCre young animals per group and n = 11–18 cCtrl and 13–18 cDatCre young animals per group; all data are represented as mean ± s.e.m.; an unpaired t-test was applied in a–d and one sample t-test in e, *P < 0.05, ***P < 0.0005.

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Figure 3 Expression analysis of DA neuron markers and analysis of catecholamines in Lmx1a/b-ablated mice. (a) Tyrosine hydroxylase (TH) immunostaining in ventral midbrain sections of 18-month-old cLmx1a/bCtrl (cCtrl) and cLmx1a/bDatCre (cDatCre) mice. Scale bar, 100 µm. (b) Unbiased stereological counting of TH-immunopositive cells in the substantia nigra (SN) and the ventral tegmental area (VTA) of 18-month-old cCtrl and cDatCre mice (SN, P = 0.0129; VTA, P = 0.0152). Nissl-counterstained sections were used to count total numbers of TH-positive and TH-negative neurons (Nissl) in the SN and VTA (total) (SN, P = 0.0022; VTA, P = 0.0260). Numbers correspond to the estimated cell numbers in one hemisphere. n = 6 animals per genotype. (c) Representative electron micrographs of TH-positive neurons from SN of 3-month- old cCtrl and cDatCre mice. The two right panels show dark degenerating neurons. n = 2 animals per genotype. Scale bar, 5 µm. (d) TH immunostaining in striatal sections of 2-month-old cCtrl and cDatCre mice. Scale bar, 100 µm. (e) Optical densitometry from TH (top) and DAT (bottom) immunostained terminals from the dorsal and ventral striatum of young (2-month-old) and old (18-month-old) cCtrl and cDatCre mice, n = 6 animals per genotype. Data are shown in arbitrary units (AU). (TH dorsal young, P = 0.0190; TH ventral young, P = 0.0381; TH dorsal old, P = 0.0175; TH ventral old P = 0.4762; DAT dorsal young, P = 0.4762; DAT ventral young, P = 0.4762; DAT dorsal old, P = 0.0111; DAT ventral old, P = 0.0012). (f) HPLC measurements of DA and the DA metabolites DOPAC and HVA in 9-month-old cCtrl and cDatCre mice. Separate analyses were performed on tissue extracts from prefrontal cortex (PFC; DA, P = 0.0058; DOPAC, P = 0.0026; HVA, P = 0.0031), hippocampus (Hip; DA, P = 1.0000; DOPAC, P = 0.0321; HVA, P = 0.0426), dorsal striatum (Striatum; DA, P = 0.0003; DOPAC, P = 0.0031; HVA, P = 0.0156) and nucleus accumbens (NAcc; DA, P = 0.0031; DOPAC, P = 0.0002; HVA, P = 0.0378). n = 10 cCtrl and 13 cDatCre animals. (g) Normal basal synaptic transmission as shown in the input-output curves obtained in the CA1 area following collateral stimulation in hippocampal slices from cCtrl and cDatCre adult mice (6 months old). (h) Representative traces and summary of long-term potentiation (LTP) experiments in cCtrl and cDatCre mice (left) and quantification of LTP induction and maintenance (right; induction, P = 0.0400; maintenance, P = 0.0022). n = 9 or 10 slices, 5 animals per genotype. All data are represented as mean ± s.e.m.; Mann-Whitney test; *P < 0.05, **P < 0.005, ***P < 0.0005.

Abnormal nerve terminals in Lmx1a/b-ablated miceWe next investigated the morphology of axonal terminals in young (3-month-old) Lmx1a/b-ablated animals. We used TH immu-noperoxidase with 3,3′-diaminobenzidine to visualize mDA nerve terminals in the striatum (Fig. 4a,b). Quantification revealed about 50% reduction in the density of TH-immunopositive profiles in Lmx1a/b-ablated mice (cLmx1a/bCtrl: 17.49 ± 1.77; cLmx1a/bDatCre: 8.33 ± 1.14; mean ± s.e.m., n = 6 animals per genotype, Mann-Whitney test P < 0.005). Notably, we found abnormally large profiles that reached up to 22 µm in diameter frequently throughout the dorsal and ventral striatum in cLmx1a/bDatCre and cLmx1bDatCre mice,

but not in cLmx1aDatCre mice, thus indicating that Lmx1b is essen-tial to maintaining normal morphology and density of mDA nerve terminals (Fig. 4a–c and Supplementary Fig. 3).

Electron microscopy studies revealed that large TH-immunoposi-tive profiles corresponded to abnormally enlarged nerve terminals (Fig. 4d–h). These enlarged presynaptic boutons, analyzed and quan-tified in serial ultrathin sections, displayed fewer synaptic vesicles at active zones (cLmx1a/bCtrl: 49.80 ± 4.08 and cLmx1a/bDatCre: 16.90 ± 3.71, mean ± s.e.m., n = 5 active zones in 2 animals per genotype, Mann-Whitney test P < 0.0005) and were filled with vacuoles and mul-tilamellar autophagic-lysosomal vesicles (ALVs) that in some instances

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contained mitochondria (Fig. 4d–h; cLmx1a/bCtrl: 0.09 ± 0.09 and cLmx1a/bDatCre: 4.06 ± 0.44, mean ± s.e.m., n = 5 active zones in 2 animals per genotype, Mann-Whitney test P < 0.0005). In agreement with these observations, antibodies against Bassoon showed a 23% lower occurrence of synaptic active zones and poor overlap of staining with synaptic vesicle markers such as VMAT2 (Slc18a2) and synaptic membrane proteins such as DAT (Supplementary Fig. 4). Together, these data clearly indicate that the synaptic morphology is disrupted in presynaptic mDA neuron terminals in Lmx1a/b-ablated mice.

Lmx1a/b ablation leads to early striatal pathologyTo determine whether the observed striatal pathology occurred because of abnormal mDA neuron development or as an acute pathologic process resulting from Lmx1a/b ablation, we investigated the consequences of Lmx1a/b ablation in adult mDA neurons in double homozygous floxed mice also harboring a copy of the tamoxifen-inducible Cre (CreERT2) under the control of Slc6a3 (DAT) gene regulatory sequences (referred to as cLmx1a/bDatCreERT2 mice). We tamoxifen-treated animals at 4 weeks of age for 5 consecutive days and analyzed them at defined time points5. Double floxed (cLmx1a/b) animals lacking the DAT-CreERT2 allele and treated with tamoxifen were used as controls (cLmx1a/bCtrl).

We found large TH-immunopositive profiles in mature neu-rons 4 weeks after ablation in the striatum of cLmx1a/bDatCreERT2 and cLmx1bDatCreERT2 mice, but not in cLmx1aDatCreERT2 mice (Supplementary Fig. 5). As was seen after Lmx1a/b ablation in embryonic postmitotic mDA neurons, electron microscopy analyses and quantifications in serial ultrathin sections confirmed that these abnormally enlarged nerve terminals displayed fewer synaptic vesi-cles at active zones and a dramatic increase in the number of ALVs

(Fig. 5a–f and Supplementary Fig. 5). DA and metabolites were also clearly diminished after adult ablation in cLmx1a/bDatCreERT2 mice (Fig. 5g). However, behavioral tests in mice 6 months after tamoxifen treatment showed a significant impairment only in the wooden block test (Fig. 5h and Supplementary Fig. 5). Striatal expression of DAT was significantly reduced in the ventral striatum 18 months after tamoxifen treatment. However, we noted no significant loss of TH and DAT expression in the dorsal striatum and no loss of TH- positive cells at this time point in cLmx1a/bDatCreERT2 animals (Fig. 5i,j). Thus, after ablation of Lmx1a/b in mature mDA neurons, a striking pathology that appears associated with autophagic-lysosomal pathway (ALP) impairment develops in striatal nerve terminals.

The ALP is impaired in Lmx1a/b-ablated miceThe ALP is central for the degradation of defective proteins and organelles and is also essential in presynaptic mDA neuron function28,29. The abnormal appearance of autophagic-lysosomal vesicles in mDA nerve terminals of Lmx1a/b-ablated mice suggested an abnormal ALP. We therefore analyzed levels of ALP components in western blots from striatal total protein extracts. Notably, proteins such as beclin1, p62, LC3BI, LC3BII, Lamp1, Lamp2 and cathepsin D were significantly decreased in cLmx1a/bDatCre and cLmx1a/bDatCreERT2 mice (Fig. 6a and Supplementary Fig. 6).

No accumulation of autophagic-lysosomal vesicles was evident in substantia nigra cell bodies; however, the number of lipofuscin granules (LPGs), requiring normal lysosomal function for their formation, were significantly reduced in cLmx1a/bDatCre mice (Fig. 6). In addition, we found electron-dense protein aggregates frequently in Lmx1a/b-ablated mice but never in control mice (Fig. 6c,d). These observations are additional indications of

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Figure 4 Abnormal morphology of DA nerve terminals in Lmx1a/b-ablated mice. (a,b) TH immunostaining in striatal sections of 2-month-old cLmx1a/bCtrl (cCtrl) and cLmx1a/bDatCre (cDatCre) mice showing nerve terminals and abnormally large profiles. Scale bar, 50 µm. (c) Area distribution of TH-immunopositive nerve terminals as a percentage of the total number of nerve terminals in 2-month-old cCtrl and cDatCre mice. n = 6 animals per genotype. (d–h) Electron micrographs of TH-labeled nerve terminals from striatum in 3-month-old cCtrl (d) and large axonal boutons from striatum in cDatCre mice (e–h). Red lines delineate borders of giant nerve terminals. f and inset in g show boxed areas around synaptic active zones (arrowheads) in e and main panel of g, respectively, at higher magnification. f and h show an area from a giant nerve terminal at higher magnification, illustrating an accumulation of autolysosomes (AL), endosome-like profiles (e) and autophagosomes (AP) in the axoplasm (ax). d, dendritic shaft; SV, synaptic vesicles; MVB, multivesicular body; m, mitochondrion. Scale bars: for d, f and h, 0.5 µm; for e and g, 1 µm. All data are represented as mean ± s.e.m.; Mann-Whitney test *P < 0.05; one-sample t-test #P ≤ 0.05.

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Figure 5 Abnormal morphology of DA nerve terminals and analysis of catecholamines, behavior and DA markers after Lmx1a/b ablation. (a–f) Electron micrographs of TH-labeled nerve terminals (ax) from striatum in cLmx1a/bCtrl (cCtrl) mice (a) and large axonal boutons (b,c) from striatum in cLmx1a/bDatCreERT2 (cERT2) mice 3 months after tamoxifen (TAM) treatment. Red lines delineate borders of giant nerve terminals. Arrowheads indicate synaptic active zones. Higher magnification micrographs (d–f) illustrating a dramatic increase in number of endosome-like structures (e), autolysosomes (AL) and autophagosomes (AP). S, soma of a postsynaptic neuron; MVB, multivesicular body; m, mitochondrion; d, dendritic shaft. Scale bars: for a–c in c, 1 µm; for d–f in f, 0.2 µm. n = 2 animals per genotype. (g) HPLC measurements of DA, DOPAC and HVA in cCtrl and cERT2 mice 9 months after TAM treatment. Separate analyses were performed on tissue extracts from olfactory bulb (OlfB; DA, P = 0.9024; DOPAC, P = 1.0000; HVA, P = 0.4470), prefrontal cortex (PFC; DA, P = 0.0494; DOPAC, P = 0.2697; HVA, P = 0.2701), dorsal striatum (Striatum; DA, P = 0.0041; DOPAC, P = 0.0266; HVA, P = 0.4002), nucleus accumbens (NAcc; DA, P = 0.0172; DOPAC, P = 0.0030; HVA, P = 0.0070) and hippocampus (Hip; DA, P = 0.3911; DOPAC, P = 0.4122; HVA, P = 0.6607). n = 9 cCtrl and 10 cERT2 animals per genotype. (h) Wooden block test in cERT2 mice 6 months after TAM treatment. Investigatory index calculated as in Figure 2d. n = 14 cCtrl and 16 cERT2 animals per group (P = 0.0095). (i) Optical densitometry from TH and DAT immunostained terminals from the dorsal and ventral striatum 18 months after TAM treatment. n = 7 animals per genotype (dorsal TH, P = 0.7104; ventral TH, P = 0.5350; dorsal DAT, P = 0.2593; ventral DAT, P = 0.0350). (j) Unbiased stereological counting of TH-immunopositive cells in the SN (P = 0.6200) and the VTA (P = 0.1248) 18 months after TAM treatment. n = 7 animals per genotype. All data are represented as mean ± s.e.m.; ns, not significant; Mann-Whitney test except in h, where an unpaired t-test was applied; *P < 0.05, **P < 0.005.

lysosomal pathway dysfunction and suggest that protein degrada-tion pathways are impaired in both distal and proximal regions of mDA neurons. We observed the most severe effects in nerve termi-nals, suggesting that the primary cellular abnormality originates in this cellular compartment.

The ALP is negatively regulated by the mammalian target of rapamycin (mTOR) signaling pathway30. Thus, the mTOR inhibi-tor rapamycin increases expression of ALP components and stimu-lates lysosomal biogenesis, and has also been shown to attenuate mDA neurodegeneration in rodents31. To test whether rapamycin could compensate for Lmx1a/b deficiency, we injected rapamycin for 10 consecutive days in young (2-month-old) cLmx1a/bDatCre mice. mTOR phosphorylation levels and increased levels of ALP-related proteins confirmed inhibition of mTOR activity (Fig. 6e,f and Supplementary Fig. 6). Rapamycin almost completely normalized the reduced striatal TH innervation (see Fig. 3) and sig-nificantly alleviated the occurrence of abnormally large TH-positive boutons in the striatum (Fig. 4) of cLmx1a/bDatCre mice (Fig. 6g,h). Thus, these results strongly indicate that a deficient ALP contributes critically to the Lmx1a/b ablation phenotype and show that a func-tional ALP is important in maintaining proper mDA projections to the striatum.

Lmx1b-mediated transcriptional regulation of ALPAs shown above (Supplementary Figs. 3 and 5), Lmx1b but not Lmx1a ablation was responsible for the described phenotype. We investigated whether Lmx1b regulated ALP genes at the tran-scriptional level by RT-qPCR analysis of mRNA isolated from E14.5 ventral midbrain from control and cLmx1a/bDatCre animals, a time point soon after Lmx1a/b ablation. We observed a robust downregula-tion of ALP genes in ablated mice (Fig. 7a). Moreover, transcription factors that are known to regulate ALP gene expression were also affected, including Ppargc1a and Tfeb, the latter encoding the tran-scription factor TFEB and reported to be a master regulator of several genes involved in lysosomal biogenesis (Fig. 7b). In con-trast, expression of key dopaminergic transcription factors was not affected in cLmx1a/bDatCre embryos, with the exception of Nurr1 (Supplementary Fig. 7). Consistent with the reduced expression of Nurr1, we also observed a reduction in expression of Th, Slc6a3 and Slc18a2 (Supplementary Fig. 7).

To further explore the ability of Lmx1b to regulate ALP gene expres-sion, we used cultured primary neurons isolated from E13.5 mouse ventral midbrain in a gain-of-function experiment. Transduction of primary neurons with an Lmx1b lentiviral vector was followed by culturing for 6 d and analysis by immunofluorescence and RT-qPCR

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(Supplementary Fig. 7). Overexpression of Lmx1b led to increased expression of most ALP genes analyzed (Fig. 7c). Reciprocally, and in accordance with observations in knockout embryos, downregula-tion of Lmx1b by using a lentiviral vector with a short hairpin RNA directed against Lmx1b (Supplementary Fig. 7) resulted in reduced expression of ALP genes (Fig. 7c). Lentiviral overexpression of Lmx1a did not affect ALP genes (Supplementary Fig. 7), again in accordance with our data showing that Lmx1b is the main contributor to the phe-notype. In conclusion, both loss- and gain-of-function experiments provide strong indications that Lmx1b transcriptionally regulates ALP genes in developing and mature mDA neurons.

Axonal transport impairment is an early feature of neurode-generation in PD, and transport deficit can contribute to axonal dystrophy (reviewed in ref. 45). We therefore analyzed the expression of genes involved in microtubule-dependent transport (Map1b), dynein-dependent retrograde transport (Dync1h1, Dctn1), and kinesin-dependent anterograde transport (Kif1a, Klc1) by RT-qPCR. cLmx1a/bDatCre animals showed a significant reduction in the expres-sion of such genes compared to cLmx1a/bCtrl animals (Fig. 7d).

The Lmx1b transcriptional regulation of these genes was confirmed by loss- and gain-of-function experiments in cultured mDA neu-rons (Fig. 7e). Our data thus suggest that Lmx1b is essential for the maintenance of the ALP and intracellular transport homeostasis.

DISCUSSIONHow neurons acquire their cell type–specific characteristics under development is well understood at the molecular level for several types of neurons. However, how neurons maintain their normal identities throughout the life of an animal has remained largely unclear, and it is therefore important to elucidate how developmental transcription factors such as Lmx1a and Lmx1b contribute to mainte-nance of specific neuronal traits3,4. Understanding these mechanisms may also be important for elucidating functions that are linked to neurodegenerative and psychiatric disease. Indeed, maintenance of the normal identity of mDA neurons is likely to be important to the prevention of PD symptoms because specific mDA neuron character-istics apparently diminish in PD long before neurons actually die5,6,24. In addition, polymorphisms have been associated with PD in genes

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Figure 6 Impaired autophagic-lysosomal pathway in Lmx1a/b ablated mice. (a) Quantification of proteins by western blot analysis in striatal total cell extracts from 9-month-old cLmx1a/bCtrl (cCtrl) and cLmx1a/bDatCre (cDatCre) mice. n = 4 cCtrl and 5 cDatCre animals. CatD, cathepsin D. (Beclin1, P = 0.0286; p62, P = 0.0286; LC3BI, P = 0.0286; LC3BII, P = 0.0286; Lamp1, P = 0.0159; Lamp2, P = 0.0159; CatD, P = 0.0303). (b,c) High magnification electron micrographs of TH-labeled cell bodies in the substantia nigra from 3-month-old cLmx1a/bDatCre (cDatCre) mice, cLmx1a/bDatCreERT2 (cERT2) mice 3 months after tamoxifen (TAM) treatment, and cLmx1a/bCtrl (cCtrl) mice. Micrographs show lipofuscin granules (LFG; b) and electron-dense protein aggregates (EPA; c). Scale bars: in b, 1 µm; in c, 0.5 µm. n = 2 animals per genotype. (d) Quantitative evaluation of LFGs and EPAs per cell in the substantia nigra (SN) in cCtrl, cDatCre (LFG, P = 0.0001; EPA, P = 0.0001) and cERT2 (LFG, P = 0.7297; EPA, P = 0.0578) mice. n = 40–74 cells per genotype, 2 animals per genotype. (e) Western blot and quantification of phospho-mTOR (P-mTOR) and mTOR expression after rapamycin injection compared to vehicle injection (intraperitoneal, 5 mg per kilogram per day for 10 consecutive days before sacrifice) in 2-month-old cCtrl and cDatCre mice. n = 5 animals (lanes 1–5) per genotype. (cCtrl versus cDatCre vehicle, P = 0.0937; cCtrl versus cDatCre rapamycin, P = 0.9361; cCtrl vehicle versus rapamycin, P = 0.0080; cDatCre vehicle versus rapamycin, P = 0.0222). Molecular weights (kDa) for the protein marker (Mk) are indicated. Western blot images are cropped, but full-length blots are presented in Supplementary Figure 6. (f) Quantification of western blot analysis of ALP proteins in striatal cell extracts from vehicle- or rapamycin-treated cCtrl and cDatCre mice. n = 5 animals per genotype. Data show fold change relative to their corresponding controls (either vehicle-treated cCtrl or rapamycin-treated cCtrl) normalized against actin expression. (p62 vehicle, P = 0.0043; p62 rapamycin, P = 0.1255; LC3BI vehicle, P = 0.0381; LC3BI rapamycin, P = 0.0317; Lamp1 vehicle, P = 0.0095; Lamp1 rapamycin, P = 0.0317; CatD vehicle, P = 0.0095; CatD rapamycin, P = 0.0317). (g) Optical densitometry from TH-immunostained striatal nerve terminals of vehicle- or rapamycin-treated cCtrl and cDatCre mice. n = 5 animals per genotype. Data are shown in arbitrary units (AU). (cCtrl versus cDatCre vehicle, P = 0.0043; cCtrl versus cDatCre rapamycin, P = 0.1255; cCtrl vehicle versus rapamycin, P = 0.4206; cDatCre vehicle versus rapamycin, P = 0.0043). (h) Density quantification of TH-immunopositive nerve terminals larger than 10 µm2 in vehicle- or rapamycin-treated cCtrl and cDatCre mice. n = 5 animals per genotype. (P = 0.0389). All data are represented as mean ± s.e.m.; ns, not significant; Mann-Whitney test *P < 0.05, **P < 0.005, ***P < 0.0005; one sample t-test #P ≤ 0.05.

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encoding mDA neuron lineage-specific transcription factors8–13,32, including LMX1A and LMX1B, and reduced levels of the NURR1 (NR4A2) and PITX3 mDA neuron transcription factor mRNAs have been noted in peripheral blood cells in PD patients33,34.

If disrupted transcription factor function contributes to PD pathol-ogy, their conditional ablation in mDA neurons after the initial phase of mDA neuron specification should result in a PD-like pathology. Indeed, we now identify a critical role for Lmx1b in the maintenance of mDA neurons. In contrast, Lmx1a function appears dispensable. As shown here for Lmx1b, and previously for Nurr1 (ref. 5), both factors regulate genes encoding components for DA synthesis and neurotransmitter handling, including TH and DAT. The down-regulation of this machinery is more drastic in Nurr1- as compared to Lmx1b-ablated mDA neurons. Since Nurr1 mRNA was downregu-lated in Lmx1b-ablated mice, the effects on neurotransmitter han-dling in these animals may be a consequence of the observed partial Nurr1 downregulation, as is likely in Foxa1 and Foxa2 conditional knockout animals6.

The regulatory role of Lmx1b extends beyond the classical mDA neuron markers, as Lmx1b is critical for the maintenance of nor-mal autophagic-lysosomal and intracellular transport functions and thus for the prevention of pathology in mDA neurons. We specu-late that the importance of Lmx1b in the regulation of ALP function reflects a specific requirement for this pathway in mDA neurons, a conclusion that is supported by previous data in PD and PD models indicating that disrupted ALP function contributes to disease31,35–37. Analogously, not only typical mDA neuron identity markers but also nuclearly encoded mitochondrial genes were downregulated in Nurr1-ablated mDA neurons5. Thus, later functions of Lmx1b and Nurr1 are not a mere reflection of their key importance in establish-ing a gene expression profile characteristic of mDA neuron identity during development: they also act later to control central cellular

homeostasis that must be under tight control to prevent the emer-gence of cellular pathology.

Several recent studies have emphasized the importance of the ALP in mDA neurons and its role in α-synuclein degradation (reviewed in ref. 38). Notably, ALP components and activity are reduced in PD patients, as well as in cell and animal PD models31,35–37. In contrast, increasing ALP activity in rodent PD models by pharmacological treatment or by gene delivery can dramatically counteract neuro-degeneration, thus suggesting new therapeutic strategies31,39. In this context, it is noteworthy that a major dysfunction occurring as a result of Lmx1a/b ablation in differentiating and adult mDA neurons is a dramatic accumulation of autophagosomes and lysosomes in axonal terminals. Accumulation of electron-dense protein aggregates in mDA neuron cell bodies, decreased expression of lysosomal genes and fewer lipofuscin granules all point in a similar direction, suggest-ing that lysosomal dysfunction is a primary deficiency after Lmx1a/b ablation that may later lead to poor survival of mDA neurons, as suggested by previous studies40. Treatment with the mTOR inhibitor rapamycin normalized reduced striatal TH expression and decreased the size of abnormally enlarged mDA nerve terminals after Lmx1a/b ablation, thus providing additional evidence that a dysfunctional ALP is central to the pathology caused by Lmx1a/b ablation. Together our results suggest that an increase in ALVs in nerve terminals leads to a disruption of the presynaptic architecture and thus to impairment of the synaptic function of mDA neurons, reflected in behavioral abnormalities and defects in synaptic plasticity. Enhanced LTP in cLmx1a/bDatCre mice is likely to be associated with alterations in the homeostatic regulation of the hippocampal neural network—for example, coupled to the impaired ALP and intracellular transport functions. Increased synaptic potentiation can lead to dysfunction of fine-tuned processing of information in hippocampal circuits and ultimately result in cognitive dysfunction41–43.

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Figure 7 Lmx1b-mediated transcriptional effects on lysosome- mediated autophagy. (a,b) RT-qPCR expression analysis of ALP and transcription factor genes in dissected ventral midbrain of E14.5 cLmx1a/bCtrl (cCtrl) and cLmx1a/bDatCre (cDatCre) embryos. n = 5 embryos per genotype. (c) RT-qPCR expression analysis of ALP genes in primary cultures from dissected ventral midbrains 6 d after infection with lentiviruses expressing GFP (LV-GFP) or Lmx1b (LV-Lmx1b), and a short-hairpin RNA with a scrambled sequence (shScrmbl) or a sequence targeting Lmx1b (shLmx1b). n = 2 biological duplicates in at least 3 independent experiments. Data are normalized to their corresponding controls (either LV-GFP or shScrmbl). (d) RT-qPCR expression analysis of genes involved in autophagosome dynamics in dissected ventral midbrain of E14.5 cCtrl and cDatCre embryos. n = 5 embryos per genotype. (e) RT-qPCR expression analysis of genes involved in autophagosome dynamics in primary cultures from dissected ventral midbrains 6 d after lentiviral infection with LV-GFP or LV-Lmx1b, and shScrmbl or shLmx1b. n = 2 biological duplicates in at least 3 independent experiments. Data are normalized to their corresponding controls (either LV-GFP or shScrmbl). All data are represented as mean ± s.e.m. of the fold change normalized to Rpl19 levels; Mann-Whitney test *P < 0.05, **P < 0.005. Becn1, beclin 1; Sqstm1, p62; Map1lc3b, LC3B; Ctsb, cathepsin B; Ctsd, cathepsin D; Ppargc1a, PGC-1α.

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On the basis of evidence from human postmortem studies, func-tional neuroimaging, genetic causes of disease and neurotoxin animal models, nigrostriatal degeneration in PD patients begins in striatal mDA nerve terminals rather than in substantia nigra cell bodies32,44. ALP dysfunction also results in dystrophic axons and altered syn-aptic function, and it thus seems likely that disrupted ALP activity represents an early abnormality in PD45. This conclusion is consist-ent with the phenotype observed in the Lmx1a/b ablation model. Accordingly, when Lmx1a/b was disrupted early, in postmitotic embryonic mDA neurons, abnormal ALP function was observed along with a progressive cell loss and decreased striatal TH expression. In contrast, when Lmx1a/b was disrupted in adult mDA neurons by tamoxifen treatment, disrupted ALP occurred before decreased stri-atal TH expression and without any apparent cell loss, suggesting that the dysregulated ALP represents an early phase of dysfunc-tion in these animals. Finally, both early and late Lmx1a/b ablation models show an abnormal social olfaction behavior, supporting the hypothesis that monoamine dysfunction may contribute to some PD non-motor symptoms46, including anosmia, which is of special relevance early in the disease47. Lmx1a/b ablation should therefore represent a useful model for investigating molecular mechanisms underlying early pathology in PD.

Finally, we also found that LMX1B was expressed in adult neuromel-anin-positive neurons in the human ventral midbrain and, notably, that expression was decreased in these neurons from PD patients. Our data suggest that an ALP-related pathology may at least in part be a conse-quence of dysregulation at the level of disrupted transcription factor function. Disruption of ongoing LMX1B expression in PD could there-fore contribute to a vicious cycle whereby further pathological changes occur as a result of transcriptional dysregulation. Our findings thus emphasize the importance of understanding the role of Lmx1a/b in postmitotic mDA neurons and the relevance to these factors to PD.

METHODSMethods and any associated references are available in the online version of the paper.

Note: Any Supplementary Information and Source Data files are available in the online version of the paper.

AcknowledgmenTSWe thank T. Samuelsson, H. Lunden-Miguel and C.-Y. Leung for technical assistance, and members of the Ericson and Perlmann laboratories for discussions. We thank S.-L. Ang (National Institute for Medical Research, London), N.-G. Larsson (Max Planck Institute, Köln) and G. Schütz (DKFZ, Heidelberg) for providing mouse lines. We thank T. Beach at the Banner Sun Health Research Institute, Arizona, USA for providing postmortem human brain samples. This work was supported by funding from the European Union, Seventh Framework Programme under grant agreement mdDANeurodev, NeuroStemCell and Synsys (T.P., J.E. and O.S.), from the Swedish Strategic Research Foundation (SSF; T.P. and P.S.), from the Swedish Research Council, and from Hjärnfonden and Parkinsonfonden (O.S.). The human tissue donation program was supported by the US National Institutes of Health (U24 NS072026 and P30 AG19610), the Arizona Department of Health Services, the Arizona Biomedical Research Commission and the Michael J. Fox Foundation for Parkinson’s Research. A.L. was supported by a Marie Curie Intra-European Fellowship for Career Development.

AUTHoR conTRIBUTIonSA.L. planned all experiments, performed histological, gene expression and western blot analyses and wrote the manuscript; N.S. performed behavioral analysis and dissection of mouse brain tissue; A.N. performed primary ventral midbrain cultures and gene expression analysis; A.A. performed behavioral analysis; N.V. cloned and produced lentiviruses; J.K.J. performed stereological and western blot analyses; M.G.G. performed electrophysiological analysis; E.S. performed electron microscopy analysis; E.J. performed histological analysis; T.Y. and J.K. performed high-performance liquid chromatography; Q.D. and J.E. generated conditional

Lmx1a gene targeted mice and helped with planning; P.S. helped with analysis and planning; O.S. performed electron microscopy analysis, helped with analysis and with writing the manuscript; T.P. together with A.L. planned all experiments and wrote the manuscript.

comPeTIng FInAncIAl InTeReSTSThe authors declare no competing financial interests.

Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

1. Björklund, A. & Dunnett, S.B. Dopamine neuron systems in the brain: an update. Trends Neurosci. 30, 194–202 (2007).

2. Cheng, H.C., Ulane, C.M. & Burke, R.E. Clinical progression in Parkinson disease and the neurobiology of axons. Ann. Neurol. 67, 715–725 (2010).

3. Deneris, E.S. & Hobert, O. Maintenance of postmitotic neuronal cell identity. Nat. Neurosci. 17, 899–907 (2014).

4. Holmberg, J. & Perlmann, T. Maintaining differentiated cellular identity. Nat. Rev. Genet. 13, 429–439 (2012).

5. Kadkhodaei, B. et al. Transcription factor Nurr1 maintains fiber integrity and nuclear-encoded mitochondrial gene expression in dopamine neurons. Proc. Natl. Acad. Sci. USA 110, 2360–2365 (2013).

6. Stott, S.R. et al. Foxa1 and foxa2 are required for the maintenance of dopaminergic properties in ventral midbrain neurons at late embryonic stages. J. Neurosci. 33, 8022–8034 (2013).

7. Di Salvio, M. et al. Otx2 controls neuron subtype identity in ventral tegmental area and antagonizes vulnerability to MPTP. Nat. Neurosci. 13, 1481–1488 (2010).

8. Bergman, O. et al. Do polymorphisms in transcription factors LMX1A and LMX1B influence the risk for Parkinson’s disease? J. Neural Transm. 116, 333–338 (2009).

9. Bergman, O. et al. PITX3 polymorphism is associated with early onset Parkinson’s disease. Neurobiol. Aging 31, 114–117 (2010).

10. Fuchs, J. et al. The transcription factor PITX3 is associated with sporadic Parkinson’s disease. Neurobiol. Aging 30, 731–738 (2009).

11. Haubenberger, D. et al. Association of transcription factor polymorphisms PITX3 and EN1 with Parkinson’s disease. Neurobiol. Aging 32, 302–307 (2011).

12. Le, W.D. et al. Mutations in NR4A2 associated with familial Parkinson disease. Nat. Genet. 33, 85–89 (2003).

13. Sleiman, P.M. et al. Characterisation of a novel NR4A2 mutation in Parkinson’s disease brain. Neurosci. Lett. 457, 75–79 (2009).

14. Deng, Q. et al. Specific and integrated roles of Lmx1a, Lmx1b and Phox2a in ventral midbrain development. Development 138, 3399–3408 (2011).

15. Yan, C.H., Levesque, M., Claxton, S., Johnson, R.L. & Ang, S.L. Lmx1a and lmx1b function cooperatively to regulate proliferation, specification, and differentiation of midbrain dopaminergic progenitors. J. Neurosci. 31, 12413–12425 (2011).

16. Andersson, E. et al. Identification of intrinsic determinants of midbrain dopamine neurons. Cell 124, 393–405 (2006).

17. Chung, S. et al. Wnt1-lmx1a forms a novel autoregulatory loop and controls midbrain dopaminergic differentiation synergistically with the SHH-FoxA2 pathway. Cell Stem Cell 5, 646–658 (2009).

18. Friling, S. et al. Efficient production of mesencephalic dopamine neurons by Lmx1a expression in embryonic stem cells. Proc. Natl. Acad. Sci. USA 106, 7613–7618 (2009).

19. Sánchez-Danés, A. et al. Efficient generation of A9 midbrain dopaminergic neurons by lentiviral delivery of LMX1A in human embryonic stem cells and induced pluripotent stem cells. Hum. Gene Ther. 23, 56–69 (2012).

20. Asbreuk, C.H., Vogelaar, C.F., Hellemons, A., Smidt, M.P. & Burbach, J.P. CNS expression pattern of Lmx1b and coexpression with ptx genes suggest functional cooperativity in the development of forebrain motor control systems. Mol. Cell. Neurosci. 21, 410–420 (2002).

21. Dai, J.X., Hu, Z.L., Shi, M., Guo, C. & Ding, Y.Q. Postnatal ontogeny of the transcription factor Lmx1b in the mouse central nervous system. J. Comp. Neurol. 509, 341–355 (2008).

22. Zou, H.L. et al. Expression of the LIM-homeodomain gene Lmx1a in the postnatal mouse central nervous system. Brain Res. Bull. 78, 306–312 (2009).

23. Ekstrand, M.I. et al. Progressive parkinsonism in mice with respiratory-chain-deficient dopamine neurons. Proc. Natl. Acad. Sci. USA 104, 1325–1330 (2007).

24. Kadkhodaei, B. et al. Nurr1 is required for maintenance of maturing and adult midbrain dopamine neurons. J. Neurosci. 29, 15923–15932 (2009).

25. Hwang, D.Y. et al. 3,4-dihydroxyphenylalanine reverses the motor deficits in Pitx3-deficient aphakia mice: behavioral characterization of a novel genetic model of Parkinson’s disease. J. Neurosci. 25, 2132–2137 (2005).

26. Lisman, J.E. & Grace, A.A. The hippocampal-VTA loop: controlling the entry of information into long-term memory. Neuron 46, 703–713 (2005).

27. Bliss, T.V. & Collingridge, G.L. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361, 31–39 (1993).

28. Friedman, L.G. et al. Disrupted autophagy leads to dopaminergic axon and dendrite degeneration and promotes presynaptic accumulation of alpha-synuclein and LRRK2 in the brain. J. Neurosci. 32, 7585–7593 (2012).

29. Hernandez, D. et al. Regulation of presynaptic neurotransmission by macroautophagy. Neuron 74, 277–284 (2012).




http://www.nature.com/reprints/index.html

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a r t I C l e S

30. Bové, J., Martinez-Vicente, M. & Vila, M. Fighting neurodegeneration with rapamycin: mechanistic insights. Nat. Rev. Neurosci. 12, 437–452 (2011).

31. Dehay, B. et al. Pathogenic lysosomal depletion in Parkinson’s disease. J. Neurosci. 30, 12535–12544 (2010).

32. Burke, R.E. & O’Malley, K. Axon degeneration in Parkinson’s disease. Exp. Neurol. 246, 72–83 (2013).

33. Le, W. et al. Decreased NURR1 gene expression in patients with Parkinson’s disease. J. Neurol. Sci. 273, 29–33 (2008).

34. Liu, H. et al. Decreased NURR1 and PITX3 gene expression in Chinese patients with Parkinson’s disease. Eur. J. Neurol. 19, 870–875 (2012).

35. Chu, Y., Dodiya, H., Aebischer, P., Olanow, C.W. & Kordower, J.H. Alterations in lysosomal and proteasomal markers in Parkinson’s disease: relationship to alpha-synuclein inclusions. Neurobiol. Dis. 35, 385–398 (2009).

36. Dehay, B. et al. Loss of P-type ATPase ATP13A2/PARK9 function induces general lysosomal deficiency and leads to Parkinson disease neurodegeneration. Proc. Natl. Acad. Sci. USA 109, 9611–9616 (2012).

37. Sánchez-Danés, A. et al. Disease-specific phenotypes in dopamine neurons from human iPS-based models of genetic and sporadic Parkinson’s disease. EMBO Mol. Med. 4, 380–395 (2012).

38. Lynch-Day, M.A., Mao, K., Wang, K., Zhao, M. & Klionsky, D.J. The role of autophagy in Parkinson’s disease. Cold Spring Harb. Perspect. Med. 2, a009357 (2012).

39. Decressac, M. et al. TFEB-mediated autophagy rescues midbrain dopamine neurons from alpha-synuclein toxicity. Proc. Natl. Acsad. Sci. USA 110, E1817–E1826 (2013).

40. Kanaan, N.M., Kordower, J.H. & Collier, T.J. Age-related accumulation of Marinesco bodies and lipofuscin in rhesus monkey midbrain dopamine neurons: relevance to selective neuronal vulnerability. J. Comp. Neurol. 502, 683–700 (2007).

41. Kaksonen, M. et al. Syndecan-3-deficient mice exhibit enhanced LTP and impaired hippocampus-dependent memory. Mol. Cell. Neurosci. 21, 158–172 (2002).

42. Migaud, M. et al. Enhanced long-term potentiation and impaired learning in mice with mutant postsynaptic density-95 protein. Nature 396, 433–439 (1998).

43. Vaillend, C., Billard, J.M. & Laroche, S. Impaired long-term spatial and recognition memory and enhanced CA1 hippocampal LTP in the dystrophin-deficient Dmd(mdx) mouse. Neurobiol. Dis. 17, 10–20 (2004).

44. Kordower, J.H. et al. Disease duration and the integrity of the nigrostriatal system in Parkinson’s disease. Brain 136, 2419–2431 (2013).

45. Yang, Y., Coleman, M., Zhang, L., Zheng, X. & Yue, Z. Autophagy in axonal and dendritic degeneration. Trends Neurosci. 36, 418–428 (2013).

46. Taylor, T.N. et al. Nonmotor symptoms of Parkinson’s disease revealed in an animal model with reduced monoamine storage capacity. J. Neurosci. 29, 8103–8113 (2009).

47. Doty, R.L. Olfaction in Parkinson’s disease and related disorders. Neurobiol. Dis. 46, 527–552 (2012).

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ONLINE METHODSAnimals. The generation of conditional Lmx1a and Lmx1b gene-targeted mice and of Cre-expressing mice has been previously described14,23,48,49. Crosses between these transgenic lines generated mice that are homozygous for the conditional targeted Lmx1a and Lmx1b alleles and heterozygous for DAT-Cre (cLmx1a/bDatCre) or heterozygous for the BAC-DAT-CreERT2 allele (cLmx1a/bDatCreERT2). Littermates homozygous for Lmx1a and Lmx1b floxed alleles and wild type for the DAT-Cre mutation or harboring no copy of the BAC-DAT-CreERT2 transgene were used as controls (cLmx1a/bCtrl). DAT-Cre and BAC-DAT-CreERT2 mice were maintained in a C57Bl6 genetic background. The rest of the mice were maintained in a mixed genetic background. Similar numbers of males and females were used in the different experimental groups. Mice were kept in rooms with controlled 12-h light/dark cycles, temperature and humidity with food and water provided ad libitum. They were housed at a maximum of 4 males per cage and 6 females per cage. All animal experiments were performed with permission from Stockholm North Animal Ethics board.

Tamoxifen administration. Tamoxifen (Sigma, T-5648) was dissolved in corn oil (Sigma, C-8267) and ethanol in a 9:1 mixture at a final concentration of 10 mg/ml. New mixture was prepared every second day. Animals were intraperitoneally injected with 2 mg of tamoxifen or vehicle (9:1 corn oil and ethanol mixture) daily for 5 consecutive days.

Histological analyses. At embryonic and early neonatal stages, the mouse brains were dissected and fixed for 2.5 h and 24 h, respectively, in 4% phosphate-buffered paraformaldehyde (PFA), cryoprotected for 24–48 h in 30% sucrose at 4 °C and embedded in OCT (Sakura Finetek). For the isolation of adult brains, animals were anesthetized and perfused through the left ventricle with body-temperature phosphate-buffered saline (PBS) followed by ice-cold 4% PFA. The brains were dissected and postfixed for 6 h in the same fixative and subsequently cryoprotected for 24–48 h in 30% sucrose at 4 °C before sectioning in a sliding microtome (at 30 µm thickness) or embedded in OCT. The embedded brains were cryosectioned at a thickness of 14 µm (midbrain) or 20 µm (forebrain) onto slides (SuperFrost Plus; Menzel Gläser). Littermates were used in all compara-tive experiments. Immunohistochemistry was done essentially as described50. Primary antibodies and dilution factors were as follows: sheep anti-TH (1:1,000; P60101-150 Pel-Freeze), rabbit anti-Lmx1a (1:7,000; AB10533 Millipore), guinea pig anti-Lmx1b (1:6,000; ref. 14), rabbit anti-TH (1:1,000; P40101-0 Pel-Freeze), rat anti-DAT (1:1,000; MAB369 Millipore), guinea pig anti-VMAT2 (1:2,000; EUD2801 Acris), mouse anti-Bassoon (1:2,000; ADI-VAM-PS003-D Stressgen). Biotinylated secondary antibodies were from Jackson ImmunoResearch Laboratories (1:200; donkey anti-rabbit 711-065-152; donkey anti-rat 712-065-153) and Alexa-tagged secondary antibodies were from Molecular Probes (1:500; donkey anti–sheep IgG Alexa Fluor 633 conjugate A21100; goat anti–rabbit IgG Alexa Fluor 555 conjugate A21428; goat anti–guinea pig IgG Alexa Fluor 488 conjugate A11073; goat anti–rat IgG Alexa Fluor 488 conjugate A11006; goat anti–mouse IgG Alexa Fluor 555 conjugate A21422). Brain sections were proc-essed for in situ hybridization as described5. Section images were studied in a confocal microscope (Axio Imager M1; Zeiss, Germany) and in a bright-field microscope (Eclipse E1000M; Nikon, Japan).

electron microscopy. Three-month-old mice (n = 3 per genotype) were per-fused transcardially with 4% PFA and 0.1% glutaraldehyde (Merck) in DPBS (Gibco). Brains were dissected out and postfixed for 4 h at 4 °C in the same fixative, washed in DPBS and cut into 100-µm slices on a vibratome (Leica, Germany). Free-floating sections were blocked in 0.1% Triton X-100 and 10% donkey serum in PBS for 1 h at room temperature, incubated with primary antibody rabbit anti-TH (1:1,000; P40101-0 Pel-Freeze) in PBS and secondary donkey anti-rabbit antibodies conjugated to biotin (1:200; 711-065-152 Jackson ImmunoResearch Laboratories), and visualized using Vectastain ABC and DAB kits (Vector Laboratories). The sections were postfixed in 3% glutaraldehyde and in 1% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4), dehydrated in ethanol and embedded in Durcupan ACM resin (Fluka). In several experi-ments, sections were stained en bloc with 1% uranyl acetate in 70% ethanol. Serial ultrathin (70 or 100 nm) and semithin (2 µm) sections were cut with a diamond knife (Diatome). Ultrathin sections were collected onto formvar-coated copper grids, counterstained with 2% uranyl acetate and lead citrate, and examined in a

Tecnai 12 electron microscope (FEI) equipped with a 2kx2k TemCam-F224HD camera (TVIPS). Complete series of up to 150 ultrathin sections were used to follow morphology of cells and synaptic terminals in three dimensions.

Stereological cell counting, optical densitometry analysis, colocalization analysis and quantifications in striatal nerve terminals and electron micrographs. The total number of TH-positive neurons in the ventral mid-brain of 2- and 18-month-old mice (n = 6 animals per group) was assessed by unbiased stereological estimations using the optical fractionator method and using the image analysis computer software Stereo Investigator (version 1.8.1.1; MicroBrightField, Germany). Every sixth section covering the entire extent of the VTA and substantia nigra in one brain hemisphere was included in the counting procedure. TH-immunostained sections were counterstained with Nissl stain to allow counting of TH-negative neurons in the VTA and substantia nigra regions and estimation of the total number of neurons in those regions. The following sampling parameters were used: (1) a fixed counting frame with a width and length of 40 µm; (2) a sampling grid size of 180 × 134 µm. The counting frames were placed randomly at the intersections of the grid within the outlined structure of interest by the software. The cells in one hemisphere were counted following the unbiased sampling rule using the 100× lens and included in the measurement when they came into focus within the optical dissector. A coefficient of error of <0.10 was accepted.

Quantification of the immunoreactivity for TH, DAT and Bassoon in striatal regions (bregma + 1.34 to + 0.98) was achieved by means of optical densitometry using ImageJ software (NIH, USA). Pixel brightness was meas-ured in one randomly selected brain hemisphere and values were corrected for nonspecific background staining by subtracting values obtained from the cortex. The Pearson correlation coefficient in double immunofluorescence stainings was calculated using the toolbox JACoP with ImageJ. Three pictures from the same striatal section level were taken for each animal (n = 3 animals per group).

Quantifications of density and size of striatal dopaminergic nerve terminals were performed on cryosections (30 µm thickness) immunostained for TH as described above. Three pictures from the same striatal section level were taken for each animal (n = 6 animals per group). The number of TH-immunoposi-tive nerve terminals in each picture and the size of each terminal were obtained using ImageJ.

The number of substantia nigra cells that presented signs of neurodegeneration and the number of lipofuscin granules and electron-dense protein aggregates present in substantia nigra cells were counted in 70–90 cells on electron micro-graphs obtained from TH-immunostained midbrain vibratome sections from n = 2 animals per genotype. The number of synaptic vesicles in striatal dopamin-ergic nerve terminals was scored from five active zones in synapses bigger than 1 µm on electron micrographs obtained from TH-immunostained vibratome sections from two animals per genotype. An observer blind to the genotypes performed the measurements and quantifications.

Behavioral tests. Adult mice (6–7 months old; n = 9 cCtrl and n = 14 cDatCre; n = 14 cCtrl and n = 16 cERT2) and old mice (18 months old; n = 18 cCtrl and n = 18 cDatCre) were tested for exploratory locomotion behavior, posture control and coordination, olfaction, working memory, short-term memory, long-term memory and emotional behavior. A single cohort of randomized mice was tested at each age with a 6-d interval between behavioral tests. Mice were randomized according to genotype. All behavioral tests were performed during the light cycle by experimenters blind to the genotype. Mice were allowed to habituate to the experiment room for at least 1 h before each test. Arenas and behavioral equipment were cleaned with 70% ethanol after each test session to avoid olfactory cues. Mice were excluded from the analysis before data collection on the basis of extremely hyperactive behavior, lack of spontaneous exploration or poor health condition.

Open field. The test was performed for 5 min in a 46 cm × 46 cm arena illuminated with a 30 lux light. Video tracking was performed using a video camera mounted in the ceiling coupled to the software ANY-maze (Stoelting Co., Wooddale, IL, USA). Vertical activity (rearing), defined as the mouse rising on its hind legs, was scored manually.

Novel object recognition test. This test was performed as described previously51.

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Pole test. Mice were placed head-up on top of a vertical pole (diameter: 8 mm, height: 55 cm) and trained for 1 d to turn and descend the pole back into the cage. On the day of the test, animals performed 3 trials. The time to orient downward and the total time to turn and descend the pole were measured, with a maximum duration of 120 s.

Beam traversal test. This test was performed as previously described52.Wooden block test. This olfactory discrimination test was conducted as

previously described53.Passive avoidance. The passive avoidance test was performed as described

previously51.T-maze. The spontaneous alternation protocol was adapted from ref. 54.

Each test begun with a forced trial followed by 14 choice trials. After each com-pleted arm choice the mouse was placed back into the start arm and confined for 5 s before each new consecutive trial. One point was assigned for each choice arm alternation.

Elevated plus-maze test. The apparatus consisted of four arms elevated one meter above the ground, two with high walls and two that lacked walls. Each mouse was individually placed in the center of the apparatus and allowed to explore it for 5 min. The number of entries into the open and closed arms were measured using ANY-maze.

Sucrose consumption test. Preference for a 2% sucrose solution over water was tested as previously described55.

Forced-swim test. This is based on mice developing behavioral despair, meas-ured as inactive floating after initial swimming or climbing, and was performed as previously described56. Each test session lasted for 6 min and was videotaped and analyzed with EthoVision XT 9.0 (Noldus, Wageningen, the Netherlands).

electrophysiology. Six-month-old mice (n = 5 per genotype) were anesthetized with isoflurane and their brains were rapidly removed into ice-cold oxygenated (95% O2 and 5% CO2) artificial cerebrospinal fluid solution (aCSF) that contained (in mM) 130 NaCl, 3.5 KCl, 1.25 NaH2PO4, 24 NaHCO3, 2 CaCl2, 1.5 MgCl2 and 10 glucose, pH 7.4 (330 mOsm). Horizontal hippocampal slices (400 µm) were prepared and were incubated for at least 2 h in an interface chamber containing oxygenated aCSF. A single slice was then transferred to a submersion recording chamber, where it was continuously perfused (1.8–2.0 mL/min) with a modi-fied aCSF (2 mM CaCl2, 1 mM MgCl2) warmed to 31–32 °C. Field excitatory postsynaptic potentials (fEPSP) were recorded with an extracellular recording pipette (filled with regular aCSF) positioned in the stratum radiatum of the CA1 area. fEPSP were evoked by stimulating Schaffer collateral–commissural (SCC) axons with a bipolar concentric electrode (FHC, Bowdoin, ME, USA) placed in the CA1 stratum radiatum. The stimulus intensity was set to ~60% of the intensity that triggered population spikes and was determined empirically for each slice. Before LTP induction, we determined stimulus/response curves using a range of stimulus intensities (8–25 µA) and examined paired-pulse facilita-tion, consisting of pairs of homosynaptic stimuli separated by a short interval (25–750 ms). For LTP experiments, stimuli were applied every 60 s for at least 25 min (baseline period) and LTP was induced using three trains of high- frequency stimulation (100 pulses at 100 Hz applied at 20-s intervals). Data were normalized with respect to the mean values of fEPSP slope recorded during the last 25 min of the baseline period.

determination of monoamines and their metabolites. Nine-month-old cLmx1a/bCtrl (n = 10) and cLmx1a/bDatCre (n = 13) mice, cLmx1a/bCtrl (n = 9) and cLmx1a/bDatCreERT2 (n = 10) mice 9 months after TAM treatment, 16- to 20-month-old cLmx1aCtrl (n = 10) and cLmx1aDatCre (n = 10) mice, and 20-month-old cLmx1bCtrl (n = 10) and cLmx1bDatCre (n = 10) mice were sacri-ficed by decapitation and brains rapidly removed and frozen in dry ice–cooled isopentane. Mice were subject to behavioral testing before sacrifice. Regions of interest were then collected using a tissue punch, weighted and kept at −80 °C until further processed. Dissected brain tissue samples (1–10 mg) were mixed at a ratio of 1:10 (w/v) with 0.2 M perchloric acid including 100 µM disodium EDTA and homogenized at 0 °C in a glass-pestle microhomogenizer. After stand-ing for 10 min on ice, the homogenates were centrifuged for 15 min at 3,000g at 4 °C. The supernatants were carefully aspirated and mixed with 0.4 M sodium acetate buffer, pH 3, at a ratio 1:2 (v/v) and filtered through a 0.22-µm centrifu-gal filter for 4 min at 14,000g at 4 °C. The filtrates were stored at −80 °C before

high-performance liquid chromatography (HPLC) analysis. The monoamine DA and its acidic metabolites DOPAC and HVA were determined by HPLC with electrochemical detection as described57. The data were recorded and integrated using a computerized data acquisition system (DataApex, Prague, Czech Republic). The limit of detection (defined as signal-to-noise ratio > 2) was 10 fmol/10 µl for each of the three monoamines.

Analysis of protein expression by western blot. Animals (n = 4 or 5 per group) were sacrificed and brains were rapidly removed and snap-frozen on dry ice–cooled isopentane. Regions of interest were collected using a tissue punch and whole cell extracts were prepared in sodium dodecyl sulfate (SDS) buffer (25 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1% SDS, 10 mM sodium pyrophosphate, 20 mM β-glycerol phosphate, 2 mM sodium orthovanadate and a protease inhibitor cocktail (Roche Applied Science)). Protein concentration was deter-mined using the BCA Protein Assay Kit (Thermo Scientific). 20–30 µg of protein were resolved by SDS-PAGE and transferred onto a nitrocellulose membrane (Invitrogen). Membranes were blocked for 1 h at room temperature in 10% skim milk in TBS plus 0.1% Tween-20 solution (TBS-T) and incubated overnight at 4 °C with the corresponding primary antibody diluted in 5% skimmed milk in TBS-T, except when using antibodies to phosphorylated proteins, in which case BSA replaced skim milk for both blocking and antibody incubation. Primary antibodies: rabbit anti beclin-1 (1:2,000, NB-500-249 Novus Biologicals), rab-bit anti-p62 (1:1,000, P0067 Sigma), rabbit anti-LC3BI-II (1:2,000, NB100-2220 Novus Biologicals), rabbit anti-Lamp1 (1:1,000, ab24170 Abcam), rabbit anti-Lamp2 (1:1,000, ab37024 Abcam), rabbit anti-cathepsin D (1:500, NBP1-42046 Novus Biologicals), rabbit anti-phospho-mTOR Ser2448 (1:1,000, 2971S Cell Signaling), rabbit anti-mTOR (1:1,000, 2983P Cell Signaling) and rabbit anti-actin (1:5,000, A2066 Sigma). After washing with TBS-T, membranes were incubated for 40 min at room temperature with a goat anti–rabbit IgG conjugated to horse-radish peroxidase (1:10,000; 31460 Thermo Scientific) diluted in 0.5% skim milk in TBS-T. Detection was by enhanced chemiluminescence with Amersham ECL western blotting detection reagents (GE Healthcare). Chemiluminescence was determined with an Image Quant LAS-4000Mini image analyzer (GE Healthcare – Fuji Photo Film). Band intensities were quantified by densitometry using Image Quant TL software (GE Healthcare, UK). An observer blind to the genotypes performed the quantifications.

Primary ventral mesencephalic cultures and lentiviral transductions. Ventral mesencephalic cells were obtained from timed-mating embryos at E13.5 as previ-ously described58. Briefly, after determining cell viability using the trypan blue dye exclusion assay (Sigma), cells were seeded at a density of 100,000 cells per well in 96-well plates on polyornithine (Sigma)- and laminin (Sigma)-coated multiwell plates. The freshly prepared cell suspensions were cultured in adhesion medium consisting of 1:1 DMEM/F12 medium (Gibco) and Neurobasal medium (Gibco), 3% FCS (PAA), 20 ng/ml fibroblast growth factor-2 (Preprotech), 1× B27 (Gibco), 1× N2 (Gibco), 1 mM sodium pyruvate, 0.25% (wt/vol) bovine serum albumin (BSA, Sigma) and 2 mM glutamine (Gibco). After 24 h, the adhesion medium was replaced with differentiation medium containing 1:1 DMEM/F12 and Neurobasal media, 0.25% (w/v) BSA, 1× B27, 1× N2, 1% FCS, 100 µM ascorbic acid (Sigma-Aldrich), 2 mM glutamine, 20 ng/ml recombinant human BDNF (R&D Systems) and 10 ng/ml recombinant human GDNF (R&D Systems).

Lentiviral MISSION shRNAs were purchased from Sigma-Aldrich (Lmx1b shRNAs: TRCN0000070594 and TRCN0000070593). The lentiviral expres-sion vector pRRL SIN.cPPT.PGK-GFP.WPRE was purchased from Addgene (USA). The lentiviral RFP vector (pLenty-III-RenLuc-RFP) was purchased from Abmgood (Richmond, CA). The mouse Lmx1b cDNA (IMAGE clone 40129984) and mouse Lmx1a cDNA (IMAGE clone 40048180) were purchased from Source Bioscience (UK) and cloned into the pRRL SIN vector downstream the PGK promoter after removal of the GFP cDNA. In-house lentiviral production was done as described59. Adherent primary cultures were transduced with the virus (MOI = 10) 24 h after seeding in differentiation medium. Cells were infected with virus over a period of 48 h and analyzed 6 d after infection for immunofluores-cence and RT-qPCR.

For immunofluorescence analysis, cells were fixed in 4% PFA 15 min at room temperature, incubated for 1 h at room temperature in blocking solution (10% normal donkey serum and 0.3% Triton X-100 in PBS) and incubated overnight

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at 4 °C with the corresponding antibodies: mouse anti-TH (1:500; MAB318 Millipore), rabbit anti-Lmx1a (1:4,000; AB10533 Millipore) and guinea pig anti-Lmx1b (1:6,000)14. Pictures were taken with an epifluorescence micro-scope (AxioImager.M1; Zeiss, Germany) equipped with a LSM 5 Exciter camera (Zeiss, Germany).

RnA extraction and gene expression analysis by RT-qPcR. Total RNA was extracted from primary ventral midbrain cultures using RNeasy Micro Kit (Qiagen) from two independent wells and three independent experiments per condition (each experiment corresponding to embryos obtained from one lit-ter). Ventral midbrain was dissected from timed mated cLmx1a/bDatCre mice at embryonic stage E14.5 and immediately snap frozen on dry ice (at least n = 4 embryos per genotype). Adult animals (n = 5 per group) were sacrificed and brains rapidly removed and snap-frozen with dry ice–cooled isopentane. Regions of interest were collected using a tissue punch. Total RNA from mouse tissue was isolated using Trizol reagent (Ambion) according to the supplier’s recommenda-tions. RNA concentration was determined using the NanoDrop assay (Thermo Scientific) and RNA integrity was assessed by running the samples on an Agilent RNA 6000 Nano chip on an Agilent 2100 BioAnalyzer (Agilent Technologies). One microgram of RNA was used for reverse transcription with oligo(dT)12–18 primers (Invitrogen) and SuperScriptIII reverse transcriptase (Invitrogen). A complete list of the Taqman gene expression assays (Applied Biosystems) is pro-vided in Supplementary Table 2. Quantitative real-time PCR was performed with Taqman Gene Expression Master Mix (Applied Biosystems) using standard procedures. Fold change was calculated with the ∆∆Ct-method and normalized to Rpl19 gene expression.

Study of human postmortem tissue. Midbrain free-floating cryosections (50 µm) from PD patients (n = 3) and age-matched control individuals without any reported neurodegenerative disease (n = 3) were provided by the Banner Sun Health Research Institute Brain and Body Donation Program of Sun City, Arizona, USA60, where all human subjects donate after signed informed written consent (Western Institutional Review Committee (WIRB), Seattle, Washington). For histological analysis, antigen retrieval was performed by a 20-min incubation in Target Retrieval Solution (DAKO) followed by incubation in formic acid for 3 min. Endogenous peroxidases were blocked for 20 min in a solution of 1% H2O2 in PBS, followed by preincubation for 1 h in blocking solution containing 10% normal donkey serum and 0.3% Triton X-100 in PBS. Rabbit polyclonal primary antibodies against human LMX1B (1:100, AP8935c Abgent) and against human LMX1A (1:200, HPA030088 Sigma) were diluted in blocking solution and applied for 48 h at 4 °C. After rinses with PBS, biotinylated secondary donkey anti-rabbit antibody (1:200; 711-065-152 Jackson ImmunoResearch Laboratories) diluted in blocking solution was applied for 1–2 h at room temperature. Biotinylated secondary antibody was followed by incubation with streptavidin–horseradish peroxidase complex (ABC kit, Vector Laboratories) for 1 h and subsequent expo-sure to the blue substrate VSG (Vector Laboratories) to visualize the immunos-taining. Identification of dopamine neurons was ascertained by the visualization of neuromelanin pigments. Quantification of the immunoreactivity for LMX1B in neuromelanin-positive cells was achieved by means of optical densitometry using ImageJ. Pixel brightness was measured in all individual neuromelanin-positive cells of four randomly selected images, and the measured values were corrected for nonspecific background staining by subtracting values obtained from the neuropil in the same images.

Statistical analysis. Statistical comparisons were performed using GraphPad Prism Software (San Diego, CA) with a non-parametric two-tailed Mann-Whitney test, parametric two-tailed unpaired t-test, one-sample two-tailed t-test or two-way ANOVA followed by Bonferroni multiple comparisons test, as indicated in the figure legends. Tests were two-sided. No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those reported in previous publications5,16. In all experiments in which a small number of mice/samples were compared (2–7 mice per genotype or 6 primary cell cul-ture preparations per condition), a nonparametric Mann-Whitney test was used because a Gaussian distribution could not be assumed considering sample size. Unpaired parametric t-tests were used only for behavior experiments where a larger number of animals were used (9–18 mice per genotype) and normality was formally tested with an F test to compare variances. A one-sample t-test was used when comparing the mean value with a hypothetical value (in the novel object recognition test, the value for the control group was equal to zero, so the mean was compared to an hypothetical mean value of 0). A two-way ANOVA was used in the passive avoidance test, where the response was affected by two factors. All data are expressed as the mean ± s.e.m., and error bars represent sem. Differences were considered significant at #P ≤ 0.05, *P < 0.05; **P < 0.005, ***P < 0.0005.

A Supplementary methods checklist is available.

48. Engblom, D. et al. Glutamate receptors on dopamine neurons control the persistence of cocaine seeking. Neuron 59, 497–508 (2008).

49. Zhao, Z.Q. et al. Lmx1b is required for maintenance of central serotonergic neurons and mice lacking central serotonergic system exhibit normal locomotor activity. J. Neurosci. 26, 12781–12788 (2006).

50. Laguna, A. et al. The protein kinase DYRK1A regulates caspase-9-mediated apoptosis during retina development. Dev. Cell 15, 841–853 (2008).

51. Eriksson, T.M. et al. Bidirectional regulation of emotional memory by 5–HT1B receptors involves hippocampal p11. Mol. Psychiatry 18, 1096–1105 (2013).

52. Schintu, N. et al. PPAR-gamma-mediated neuroprotection in a chronic mouse model of Parkinson’s disease. Eur. J. Neurosci. 29, 954–963 (2009).

53. Tillerson, J.L. et al. Olfactory discrimination deficits in mice lacking the dopamine transporter or the D2 dopamine receptor. Behav. Brain Res. 172, 97–105 (2006).

54. Deacon, R.M. & Rawlins, J.N. T-maze alternation in the rodent. Nat. Protoc. 1, 7–12 (2006).

55. Krishnan, V. et al. Molecular adaptations underlying susceptibility and resistance to social defeat in brain reward regions. Cell 131, 391–404 (2007).

56. Cryan, J.F. et al. Use of dopamine-beta-hydroxylase-deficient mice to determine the role of norepinephrine in the mechanism of action of antidepressant drugs. J. Pharmacol. Exp. Ther. 298, 651–657 (2001).

57. Yoshitake, T., Kehr, J., Todoroki, K., Nohta, H. & Yamaguchi, M. Derivatization chemistries for determination of serotonin, norepinephrine and dopamine in brain microdialysis samples by liquid chromatography with fluorescence detection. Biomed. Chromatogr. 20, 267–281 (2006).

58. Pruszak, J., Just, L., Isacson, O. & Nikkhah, G. Isolation and culture of ventral mesencephalic precursor cells and dopaminergic neurons from rodent brains. Curr. Protoc. Stem Cell Biol. Ch. 2, unit 2D 5 (2009).

59. Malewicz, M. et al. Essential role for DNA-PK-mediated phosphorylation of NR4A nuclear orphan receptors in DNA double-strand break repair. Genes Dev. 25, 2031–2040 (2011).

60. Beach, T.G. et al. The Sun Health Research Institute Brain Donation Program: description and experience, 1987–2007. Cell Tissue Bank 9, 229–245 (2008).

Dopaminergic control of autophagic-lysosomal function implicates Lmx1b in Parkinson's disease

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