Loss of substantia nigra (SN) neurons causes Parkinson's disease. Pathological examination of a healthy patient (A) reveals typical pigmented DA neurons in the SN (arrows); in contrast, loss of SN neurons leads to pigment disappearance in the PD brain (B, arrows). Magnification of the SN area reveals a dense network of melanin-pigmented SN neurons in the healthy brain (C) while most of SN neurons are lost in PD (D). Some of the remaining neurons in PD contain insoluble cytoplasmic protein aggregates (Lewy Bodies, E) that are made of aggregated alpha-synuclein and other proteins. The melanin-containing granules have a red-brown hue and are distributed in the cytosol of all SN neurons (C-E). The picture in E is the higher magnification of the dark-boxed area in D. Adapted from Agamanolis, 2006.

Contexts in source publication

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... and molecular aspects of neurodegenerative diseases 12 Figure 2 Loss of substantia nigra neurons causes Parkinson's disease 15 Figure 3 Dopaminergic cell groups and the nigrostriatal pathway 16 Figure 4 Basal ganglia and the control of movement 18 Figure 5 Classification of major diseases that manifest with Parkinsonisn 20 Figure 6 Structure of proteins linked to Parkinson's disease 22 Figure 7 Putative functions for DJ-1 27 Figure 8 Mechanisms of cell death in Parkinson's disease 30 Figure 9 Neurotrophins signal via Trk receptors and p75NTR 37 Figure Targeted gene expression using the GAL4-UAS system 130 HD Huntington's disease HSCR Hirschsprung's disease Iba-1 ...

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... melanin pigment and the remaining SN neurons display intracytoplasmic protein aggregates containing alpha-synuclein (Lewy Bodies; Figure 2B,D,E). SN serves mainly as input to the basal ganglia circuit, by supplying the striatum with dopamine (nigrostriatal pathway; Figure 3). ...

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... mutations leading to constitutive activity of Ret have been identified in three related dominantly inherited cancer syndromes: multiple endocrine neoplasia type 2A (MEN2A), 2B (MEN2B) and familial medullary thyroid carcinoma (FMTC) ( Santoro et al., 2004). Substitution of cysteine Cys634 (located in the cysteine-rich domain at the boundary with the trans- membrane domain) with other amino acids accounts for 85 % of all MEN2A cases and leads to the formation of a covalent link between two mutated Ret MEN2A receptors (thus generating a dimer that displays constitutive kinase activity) ( Figure 12). Substitution of methionine 918 from the kinase domain of Ret with threonine (M918T) accounts for 95 % of all MEN2B cases and leads to a constitutively active, monomeric, Ret MEN2B receptor ( Santoro et al., 2004) ( Figure 12). ...

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... of cysteine Cys634 (located in the cysteine-rich domain at the boundary with the trans- membrane domain) with other amino acids accounts for 85 % of all MEN2A cases and leads to the formation of a covalent link between two mutated Ret MEN2A receptors (thus generating a dimer that displays constitutive kinase activity) ( Figure 12). Substitution of methionine 918 from the kinase domain of Ret with threonine (M918T) accounts for 95 % of all MEN2B cases and leads to a constitutively active, monomeric, Ret MEN2B receptor ( Santoro et al., 2004) ( Figure 12). ...

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... to the classical RTK paradigm, GFLs -acting as homo-dimers-recruit 2 molecules of GFRα and subsequently 2 molecules of Ret receptor ( Figure 12). Formation of the GDNF/GFRα/Ret tripartite complex promotes RET dimerization and receptor trans-autophosphorylation within the RET intracellular kinase domain leading to recruitement of phospho-tyrosine-binding adaptors that lead to activation of downstream signaling cascades ( Figure 12). ...

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... to the classical RTK paradigm, GFLs -acting as homo-dimers-recruit 2 molecules of GFRα and subsequently 2 molecules of Ret receptor ( Figure 12). Formation of the GDNF/GFRα/Ret tripartite complex promotes RET dimerization and receptor trans-autophosphorylation within the RET intracellular kinase domain leading to recruitement of phospho-tyrosine-binding adaptors that lead to activation of downstream signaling cascades ( Figure 12). ...

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... used the astrocytic marker glial fibrillary acidic protein (GFAP) to detect alterations in astrocyte number in the brains of Ret and TrkB mutant and control mice. GFAP- labeled astrocytes displayed their typical star-shape morphology ( Figure 20) and were found in several brain regions, including the cortex, the striatum or the SN (data not shown). The measurement of GFAP-immunoreactive astrocyte density in the dorsal striatum revealed a similar (low) astrocyte density in both 12-month-old DAT-Ret and control mice (Figure 20A-C; n=3 mice/group, p=0.9, Student's t-test). ...

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... labeled astrocytes displayed their typical star-shape morphology ( Figure 20) and were found in several brain regions, including the cortex, the striatum or the SN (data not shown). The measurement of GFAP-immunoreactive astrocyte density in the dorsal striatum revealed a similar (low) astrocyte density in both 12-month-old DAT-Ret and control mice (Figure 20A-C; n=3 mice/group, p=0.9, Student's t-test). Remarkably, at 24 months, a massive striatal astrogliosis was detected in DAT-Ret mice as compared to control and DAT-TrkB mice (Figure 20D control mice, despite the marked degeneration of DA neuron cell bodies in this area (Figure 20G-I; n=3 mice/group, p=0.24, ...

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... measurement of GFAP-immunoreactive astrocyte density in the dorsal striatum revealed a similar (low) astrocyte density in both 12-month-old DAT-Ret and control mice (Figure 20A-C; n=3 mice/group, p=0.9, Student's t-test). Remarkably, at 24 months, a massive striatal astrogliosis was detected in DAT-Ret mice as compared to control and DAT-TrkB mice (Figure 20D control mice, despite the marked degeneration of DA neuron cell bodies in this area (Figure 20G-I; n=3 mice/group, p=0.24, Student's t-test). ...

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... measurement of GFAP-immunoreactive astrocyte density in the dorsal striatum revealed a similar (low) astrocyte density in both 12-month-old DAT-Ret and control mice (Figure 20A-C; n=3 mice/group, p=0.9, Student's t-test). Remarkably, at 24 months, a massive striatal astrogliosis was detected in DAT-Ret mice as compared to control and DAT-TrkB mice (Figure 20D control mice, despite the marked degeneration of DA neuron cell bodies in this area (Figure 20G-I; n=3 mice/group, p=0.24, Student's t-test). ...

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... used immunohistochemistry for the Ionized binding calcium adapter molecule (Iba)- 1 to specifically label microglia in brains of DAT-Ret and control mice. Iba-1 labeling revealed the presence of microglial cells both in the striatum ( Figure 21A,B) and the SN ( Figure 21H,I) of mutant and control mice. I first used Iba-1 stained sections form the striatum of mutant and control mice and determined the density of microglial cells in this area. ...

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... used immunohistochemistry for the Ionized binding calcium adapter molecule (Iba)- 1 to specifically label microglia in brains of DAT-Ret and control mice. Iba-1 labeling revealed the presence of microglial cells both in the striatum ( Figure 21A,B) and the SN ( Figure 21H,I) of mutant and control mice. I first used Iba-1 stained sections form the striatum of mutant and control mice and determined the density of microglial cells in this area. ...

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... first used Iba-1 stained sections form the striatum of mutant and control mice and determined the density of microglial cells in this area. The density of microglial cells in the striatum is similar in 24-month-old DAT-Ret and control mice (Figure 21A-C; n=4 mice/group, p=0.065, Student's t-test), suggesting that degenerating DA fibers and post-synaptic dysfunction in DAT-Ret mice are not a strong recruiting signal for microglial cells. ...

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... then set up to determine the density of microglial cells in the SN area. For each coronal section stained for Iba-1, I used the consecutive section stained for TH, to allow precise delineation of the SN area ( Figure 21D-G ...

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... performed a series of behavioral tests that are routinely used to detect motor alterations in rodents ( Figure 22). To evaluate the balance and coordination abilities of mutant and control mice, the rotarod test and the swimming test were used; the open- field and forced swimming test allowed the analysis of both general and horizontal activity. ...

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... recording the spontaneous general activity of mutant and control mice in the open-field arena, no major differences between DAT-Ret and control animals were observed (data not shown). When analyzing the general and horizontal activity in the forced swimming test, no difference was observed between the different groups of mice ( Figure 22B). Similarly, the coordination and balance abilities as assessed in the rotarod and swimming tests were similar in aging DAT-Ret and control mice ( Figure 22A,C, n>15, p=NS, Student's t-test). ...

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... analyzing the general and horizontal activity in the forced swimming test, no difference was observed between the different groups of mice ( Figure 22B). Similarly, the coordination and balance abilities as assessed in the rotarod and swimming tests were similar in aging DAT-Ret and control mice ( Figure 22A,C, n>15, p=NS, Student's t-test). Moreover, measurements performed by Edgar Kramer also revealed that the total levels of striatal dopamine were not significantly altered in aging DAT-Ret and DAT- TrkB mice compared to controls (data not shown). ...

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... brain coronal sections immunostained for the DA marker TH ( Figure 23A-C), I determined the number of TH-positive DA neurons in the SN of 3-month-old mutant and control animals. The same number of DA neurons was found in DAT-Ret;DJ-1 double mutant and in control animals, indicating that the nigrostriatal system developed normally in the absence of Ret and DJ-1 function (Fig. 23G). ...

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... immunostained for the DA marker TH ( Figure 23A-C), I determined the number of TH-positive DA neurons in the SN of 3-month-old mutant and control animals. The same number of DA neurons was found in DAT-Ret;DJ-1 double mutant and in control animals, indicating that the nigrostriatal system developed normally in the absence of Ret and DJ-1 function (Fig. 23G). I then evaluated the number of aging (18-and 24-month-old) SN neurons in mutant and control mice ( Figure 23). I found that 18-month-old mice lacking DJ-1 function have a normal complement of SN neurons, consistent with the previously mentioned studies ( Figure 23H; DJ-1 -/-vs. CTRL p= NS, Student's t-test). DAT-Ret mice display a ...

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... same number of DA neurons was found in DAT-Ret;DJ-1 double mutant and in control animals, indicating that the nigrostriatal system developed normally in the absence of Ret and DJ-1 function (Fig. 23G). I then evaluated the number of aging (18-and 24-month-old) SN neurons in mutant and control mice ( Figure 23). I found that 18-month-old mice lacking DJ-1 function have a normal complement of SN neurons, consistent with the previously mentioned studies ( Figure 23H; DJ-1 -/-vs. ...

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... then evaluated the number of aging (18-and 24-month-old) SN neurons in mutant and control mice ( Figure 23). I found that 18-month-old mice lacking DJ-1 function have a normal complement of SN neurons, consistent with the previously mentioned studies ( Figure 23H; DJ-1 -/-vs. CTRL p= NS, Student's t-test). ...

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... p= NS, Student's t-test). DAT-Ret mice display a moderate loss of DA neurons (25 % loss), in agreement with our previous observations ( Figure 23H; n=5 mice/group, DAT-Ret vs. CTRL p<0.01 Student's t-test). Remarkably, when analyzing 18-month-old DAT-Ret/DJ-1aging mice, I found a significant decrease in the number of SN neurons (37 % loss) compared to DAT-Ret mice and DJ-1 -/-and controls mice ( Figure 23H; n=5 mice/group; DAT-Ret/DJ-1 vs. CTRL p<0.001, ...

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... mice display a moderate loss of DA neurons (25 % loss), in agreement with our previous observations ( Figure 23H; n=5 mice/group, DAT-Ret vs. CTRL p<0.01 Student's t-test). Remarkably, when analyzing 18-month-old DAT-Ret/DJ-1aging mice, I found a significant decrease in the number of SN neurons (37 % loss) compared to DAT-Ret mice and DJ-1 -/-and controls mice ( Figure 23H; n=5 mice/group; DAT-Ret/DJ-1 vs. CTRL p<0.001, DAT- Ret/DJ-1 vs. DJ-1 -/-p<0.001 ...

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... t- test). At 24-months, DAT-Ret/DJ-1 mice lost 41 % of their SN neurons, in contrast to DAT-Ret (25 % loss), DJ-1 -/-and control mice (no loss of SN neurons; Figure 23I; n=5 mice/group; DAT-Ret/DJ-1 vs. CTRL p<0.001, DAT-Ret/DJ-1 vs. DJ-1 -/- p<0.001 and DAT-Ret/DJ-1 vs. DAT-Ret p<0.01, ...

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... then used as a second, independent DA marker to rule out that the previously observed effects were due to downregulation of TH expression; I used an antibody against Pitx-3 ( Figure 23D-F), which is expressed by all differentiated DA ( Smidt et al., 2004). I found, similarly, that 18-month-old DJ-1 -/-mice have a normal complement of SN neurons and DAT-Ret mice display a moderate loss of SN neurons (25 %; Figure 23J; DJ-1 -/-vs. ...

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... then used as a second, independent DA marker to rule out that the previously observed effects were due to downregulation of TH expression; I used an antibody against Pitx-3 ( Figure 23D-F), which is expressed by all differentiated DA ( Smidt et al., 2004). I found, similarly, that 18-month-old DJ-1 -/-mice have a normal complement of SN neurons and DAT-Ret mice display a moderate loss of SN neurons (25 %; Figure 23J; DJ-1 -/-vs. CTRL p=NS; DAT-Ret vs. CTRL p<0.01 Student's t- test). ...

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... p=NS; DAT-Ret vs. CTRL p<0.01 Student's t- test). 18-month-old DAT-Ret;DJ-1 double mutant mice lost 41 % of their SN neurons, significantly more than age-matched DAT-Ret, DJ-1 -/-and control mice ( Figure 23J; n=5 mice/group; DAT-Ret/DJ-1 vs. CTRL p<0.001, DAT-Ret/DJ-1 vs. DJ-1 -/-p<0.001 ...

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... in a subpopulation of the SN. Furthermore, to exclude that the DAT-Cre transgene and the mutant DJ-1 allele somehow genetically interacted, I compared the numbers of TH-positive neurons in 18-month-old DAT-Cre;DJ-1 -/-mice, DAT-Cre transgenics and littermate controls; in all these three mutant lines, a similar number of SN neurons was found (Fig. 23K). In addition, quantifications performed by Pontus Klein revealed no loss of VTA neurons in 18-month-old DAT-Ret/DJ-1 mice as compared to controls, further suggesting that the survival dependency on Ret and DJ- 1 signaling is specific for SN neurons (data not shown). Taken together, these results indicate that DJ-1 is required for ...

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... is the GIRK2 neurons that are preferentially lost in PD ( Liang et al., 1996;Yamada et al., 1990). We asked whether both subpopulations require Ret and DJ-1 activity for survival during aging (Figure 24). ...

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... stained coronal brain sections from mutant and control mice for GIRK2 ( Figure 24A-D) and performed stereological quantifications to determine the number of GIRK2-positive SN neurons in 24-month-old animals. Removal of DJ-1 had no effect on the number of GIRK2-positive neurons ( Figure 24A,B,I). ...

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... stained coronal brain sections from mutant and control mice for GIRK2 ( Figure 24A-D) and performed stereological quantifications to determine the number of GIRK2-positive SN neurons in 24-month-old animals. Removal of DJ-1 had no effect on the number of GIRK2-positive neurons ( Figure 24A,B,I). Remarkably, while removal of Ret function alone caused a partial reduction of GIRK2-positive neurons (33% loss), combined removal of Ret and DJ-1 had the strongest effect (51% loss; p<0.001 ...

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... while removal of Ret function alone caused a partial reduction of GIRK2-positive neurons (33% loss), combined removal of Ret and DJ-1 had the strongest effect (51% loss; p<0.001 DAT-Ret/DJ-1 double vs. CTRL; p<0.01 DAT-Ret/DJ-1 double vs. DAT-Ret single mutants, Student's t-test; Figure 24C,D,I). Therefore, Ret and DJ-1 activity is required to maintain half of the GIRK2-immunoreactive DA neurons in the SN during aging. ...

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... second DA population in the SN was labeled using a Calbindin-specific antibody. Interestingly, the Calbindin-immunoreactive subpopulation in the SN was unaffected in all groups ( Figure 24E-H,J). Thus, the neuronal population that is most critically dependent on Ret and DJ-1 function is the GIRK2-positive subpopulation, while the other midbrain dopaminergic cells are grossly unaffected in the mutant mice. ...

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... next evaluated the possibility that Ret and DJ-1 cooperate in maintaining target innervation of nigral DA neurons. I labeled DA fibers in the striatum using TH as a marker and compared the fiber density in 18-and 24-months mutant and control mice (Figure 25A-D). Quantification of TH-positive fiber density confirmed a marked decrease in the dorsal striatum of 18-and 24-month-old DAT-Ret single mutants compared to age-matched controls ( Figure 25C,H,I; n= 5 mice/group; DAT-Ret vs. CTRL p<0,001, Student's t-test). ...

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... labeled DA fibers in the striatum using TH as a marker and compared the fiber density in 18-and 24-months mutant and control mice (Figure 25A-D). Quantification of TH-positive fiber density confirmed a marked decrease in the dorsal striatum of 18-and 24-month-old DAT-Ret single mutants compared to age-matched controls ( Figure 25C,H,I; n= 5 mice/group; DAT-Ret vs. CTRL p<0,001, Student's t-test). In contrast, no significant reductions in the density of DA fibers were observed in the striatum of DJ-1 single mutant mice ( Figure 25B,H,I). ...

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... of TH-positive fiber density confirmed a marked decrease in the dorsal striatum of 18-and 24-month-old DAT-Ret single mutants compared to age-matched controls ( Figure 25C,H,I; n= 5 mice/group; DAT-Ret vs. CTRL p<0,001, Student's t-test). In contrast, no significant reductions in the density of DA fibers were observed in the striatum of DJ-1 single mutant mice ( Figure 25B,H,I). Interestingly, DAT-Ret/DJ-1 double mutants displayed reductions of TH- positive fibers that were in the same range as DAT-Ret single mutants (46 % at 18- months and 52 % at 24-months, DAT-Ret/DJ-1 vs. DAT-Ret p=NS, Student's t-test; Figure 25D,H,I). ...

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... contrast, no significant reductions in the density of DA fibers were observed in the striatum of DJ-1 single mutant mice ( Figure 25B,H,I). Interestingly, DAT-Ret/DJ-1 double mutants displayed reductions of TH- positive fibers that were in the same range as DAT-Ret single mutants (46 % at 18- months and 52 % at 24-months, DAT-Ret/DJ-1 vs. DAT-Ret p=NS, Student's t-test; Figure 25D,H,I). I then used the dopamine transporter (DAT) as a second, independent marker for DA axons ( Figure 25E-G). ...

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... DAT-Ret/DJ-1 double mutants displayed reductions of TH- positive fibers that were in the same range as DAT-Ret single mutants (46 % at 18- months and 52 % at 24-months, DAT-Ret/DJ-1 vs. DAT-Ret p=NS, Student's t-test; Figure 25D,H,I). I then used the dopamine transporter (DAT) as a second, independent marker for DA axons ( Figure 25E-G). DAT-Cre knock-in mice were used as controls, since they have reduced levels of DAT protein (the Cre transgene being inserted in the 5'-UTR of the DAT gene). ...

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... knock-in mice were used as controls, since they have reduced levels of DAT protein (the Cre transgene being inserted in the 5'-UTR of the DAT gene). Both DAT-Ret and DAT-Ret/DJ-1 24-month-old mutants displayed a 54 % reduction in DAT-immunoreactive fiber density relative to age-matched DAT-Cre control mice (n =4-5 mice/group; DAT-Ret or DAT-Ret/DJ-1 vs. DAT-Cre p< 0,001 and DAT-Ret vs. DAT-Ret/DJ-1 p=NS, Student's t-test; Figure 25E-G,J;). Thus, DJ-1 activity is dispensable for long-term maintenance of DA fibers deprived of Ret- mediated trophic support. ...

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... DAT-Ret mice were found to have post-synaptic defects (Figure 19), enhanced glial cell recruitment in the dorsal striatum ( Figure 20) but no significant behavioral deficits ( Figure 22). To determine whether the increased degeneration of SN DA cell bodies in DAT-Ret/DJ-1 mice has any impact on neuroinflammatory processes in the striatum, I labeled microglial cells and astrocytes in the dorsal striatum of 18-and 24- months old control, DAT-Ret and DJ-1 -/-single and DAT-Ret/DJ-1 double mutant mice ( Figure 26). ...

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... DAT-Ret mice were found to have post-synaptic defects (Figure 19), enhanced glial cell recruitment in the dorsal striatum ( Figure 20) but no significant behavioral deficits ( Figure 22). To determine whether the increased degeneration of SN DA cell bodies in DAT-Ret/DJ-1 mice has any impact on neuroinflammatory processes in the striatum, I labeled microglial cells and astrocytes in the dorsal striatum of 18-and 24- months old control, DAT-Ret and DJ-1 -/-single and DAT-Ret/DJ-1 double mutant mice ( Figure 26). ...

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... DAT-Ret mice were found to have post-synaptic defects (Figure 19), enhanced glial cell recruitment in the dorsal striatum ( Figure 20) but no significant behavioral deficits ( Figure 22). To determine whether the increased degeneration of SN DA cell bodies in DAT-Ret/DJ-1 mice has any impact on neuroinflammatory processes in the striatum, I labeled microglial cells and astrocytes in the dorsal striatum of 18-and 24- months old control, DAT-Ret and DJ-1 -/-single and DAT-Ret/DJ-1 double mutant mice ( Figure 26). ...

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... visible differences in the density of Iba-1-immunoreactive microglial cells in the striatum were observed among the aforementioned groups of animals ( Figure 26A-D). This situation parallels the previous finding that 24-month-old DAT-Ret and control mice have similar microglial cell densities in the striatum (Figure 21A-C). ...

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... visible differences in the density of Iba-1-immunoreactive microglial cells in the striatum were observed among the aforementioned groups of animals ( Figure 26A-D). This situation parallels the previous finding that 24-month-old DAT-Ret and control mice have similar microglial cell densities in the striatum (Figure 21A-C). I then used an anti-GFAP antibody to label astrocytes in 18-and 24-months animals. ...

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... then used an anti-GFAP antibody to label astrocytes in 18-and 24-months animals. At 18- months, the density of GFAP-labeled astrocytes was similar in all mutant and control groups ( Figure 26E-H,M). At 24 months however, the astrocyte density was visibly increased in both DAT-Ret and DAT-Ret/DJ-1 mice, relative to control and DJ-1 deficient mice ( Figure 26I-L), consistent with the previously observed increase in 24- month-old DAT-Ret vs. control mice ( Figure 20D-F). ...

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... 18- months, the density of GFAP-labeled astrocytes was similar in all mutant and control groups ( Figure 26E-H,M). At 24 months however, the astrocyte density was visibly increased in both DAT-Ret and DAT-Ret/DJ-1 mice, relative to control and DJ-1 deficient mice ( Figure 26I-L), consistent with the previously observed increase in 24- month-old DAT-Ret vs. control mice ( Figure 20D-F). Quantification of astrocyte density revealed a 2-fold increase in 24-month-old DAT-Ret and DAT-Ret/DJ-1 mice relative to control and DJ-1 mutant mice, but no significant difference between DAT- Ret single and DAT-Ret/DJ-1 double mutants (n=4-5 mice/group; DAT-Ret or DAT- Ret/DJ-1 vs. CTRL p< 0,01 and DAT-Ret vs. DAT-Ret/DJ-1 p=NS, Student's t-test; Figure 26N). ...

Context 47

... 18- months, the density of GFAP-labeled astrocytes was similar in all mutant and control groups ( Figure 26E-H,M). At 24 months however, the astrocyte density was visibly increased in both DAT-Ret and DAT-Ret/DJ-1 mice, relative to control and DJ-1 deficient mice ( Figure 26I-L), consistent with the previously observed increase in 24- month-old DAT-Ret vs. control mice ( Figure 20D-F). Quantification of astrocyte density revealed a 2-fold increase in 24-month-old DAT-Ret and DAT-Ret/DJ-1 mice relative to control and DJ-1 mutant mice, but no significant difference between DAT- Ret single and DAT-Ret/DJ-1 double mutants (n=4-5 mice/group; DAT-Ret or DAT- Ret/DJ-1 vs. CTRL p< 0,01 and DAT-Ret vs. DAT-Ret/DJ-1 p=NS, Student's t-test; Figure 26N). ...

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... 24 months however, the astrocyte density was visibly increased in both DAT-Ret and DAT-Ret/DJ-1 mice, relative to control and DJ-1 deficient mice ( Figure 26I-L), consistent with the previously observed increase in 24- month-old DAT-Ret vs. control mice ( Figure 20D-F). Quantification of astrocyte density revealed a 2-fold increase in 24-month-old DAT-Ret and DAT-Ret/DJ-1 mice relative to control and DJ-1 mutant mice, but no significant difference between DAT- Ret single and DAT-Ret/DJ-1 double mutants (n=4-5 mice/group; DAT-Ret or DAT- Ret/DJ-1 vs. CTRL p< 0,01 and DAT-Ret vs. DAT-Ret/DJ-1 p=NS, Student's t-test; Figure 26N). Therefore, combined deletion of Ret and DJ-1 does not accelerate the striatal neuroinflammatory processes observed in aging DAT-Ret deficient mice. ...

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... understand whether absence of Ret and DJ-1 function affects the DA neurotransmission in the striatum we performed, together with Pontus Klein, open- field analysis on 18-month-old mutant and control mice ( Figure 26O). Mice were tested, without previous training, in an open-field arena and their horizontal activity was measured during two consecutive sessions. ...

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... previous reports Goldberg et al., 2005) we found that DJ-1 deficient mice were moderately hypoactive as compared to controls carrying floxed alleles of Ret or heterozygous Ret-mutants (n=7-16 mice/group and DJ-1 -/-vs. CTRL p<0,01 Student's t-test; Figure 26O). In contrast, mice carrying the DAT-Cre transgene were found to be hyperactive (CTRL vs. DAT-Cre p<0.01, ...

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... contrast, mice carrying the DAT-Cre transgene were found to be hyperactive (CTRL vs. DAT-Cre p<0.01, Student's t-test; Figure 26O); this is likely due to reduced levels of dopamine transporter, caused by the insertion of the Cre transgene in the DAT 5'-UTR (data not shown). Reduced levels of dopamine transporter allow more dopamine to accumulate at the synapse, as a result of decreased reuptake by DA presynaptic terminals; indeed, mice that partially or completely lack the dopamine transporter are hyperactive and display more dopamine in the striatum ( Perona et al., 2008) ...

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... first confirmed that mutant α-synA30P shows abnormal accumulation in SN cell bodies. Using an antibody that recognizes both the endogenous and the human transgenic α-syn, I found that endogenous α-syn is located at axon terminals in control animals ( Figure 27C), and no protein is detected in SN cell bodies ( Figure 27A). In contrast, transgenic α-syn Ala30Pro localizes at axon terminals ( Figure 27, compare D with C) but also accumulates in the SN cell body ( Figure 27B). ...

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... first confirmed that mutant α-synA30P shows abnormal accumulation in SN cell bodies. Using an antibody that recognizes both the endogenous and the human transgenic α-syn, I found that endogenous α-syn is located at axon terminals in control animals ( Figure 27C), and no protein is detected in SN cell bodies ( Figure 27A). In contrast, transgenic α-syn Ala30Pro localizes at axon terminals ( Figure 27, compare D with C) but also accumulates in the SN cell body ( Figure 27B). ...

Context 54

... an antibody that recognizes both the endogenous and the human transgenic α-syn, I found that endogenous α-syn is located at axon terminals in control animals ( Figure 27C), and no protein is detected in SN cell bodies ( Figure 27A). In contrast, transgenic α-syn Ala30Pro localizes at axon terminals ( Figure 27, compare D with C) but also accumulates in the SN cell body ( Figure 27B). The same conclusion was reached using a human specific α-syn antibody; the transgenic protein localizes both to the striatum ( Figure 27H) and SN cell body ( Figure 27F), while the endogenous α-syn is not detected by the human-specific antibody ( Figure 27E,G). ...

Context 55

... an antibody that recognizes both the endogenous and the human transgenic α-syn, I found that endogenous α-syn is located at axon terminals in control animals ( Figure 27C), and no protein is detected in SN cell bodies ( Figure 27A). In contrast, transgenic α-syn Ala30Pro localizes at axon terminals ( Figure 27, compare D with C) but also accumulates in the SN cell body ( Figure 27B). The same conclusion was reached using a human specific α-syn antibody; the transgenic protein localizes both to the striatum ( Figure 27H) and SN cell body ( Figure 27F), while the endogenous α-syn is not detected by the human-specific antibody ( Figure 27E,G). ...

Context 56

... contrast, transgenic α-syn Ala30Pro localizes at axon terminals ( Figure 27, compare D with C) but also accumulates in the SN cell body ( Figure 27B). The same conclusion was reached using a human specific α-syn antibody; the transgenic protein localizes both to the striatum ( Figure 27H) and SN cell body ( Figure 27F), while the endogenous α-syn is not detected by the human-specific antibody ( Figure 27E,G). Thus, overexpressed mutant Ala30Pro α-syn shows abnormal accumulation in the SN cell body and is a potential source of cellular stress. ...

Context 57

... contrast, transgenic α-syn Ala30Pro localizes at axon terminals ( Figure 27, compare D with C) but also accumulates in the SN cell body ( Figure 27B). The same conclusion was reached using a human specific α-syn antibody; the transgenic protein localizes both to the striatum ( Figure 27H) and SN cell body ( Figure 27F), while the endogenous α-syn is not detected by the human-specific antibody ( Figure 27E,G). Thus, overexpressed mutant Ala30Pro α-syn shows abnormal accumulation in the SN cell body and is a potential source of cellular stress. ...

Context 58

... contrast, transgenic α-syn Ala30Pro localizes at axon terminals ( Figure 27, compare D with C) but also accumulates in the SN cell body ( Figure 27B). The same conclusion was reached using a human specific α-syn antibody; the transgenic protein localizes both to the striatum ( Figure 27H) and SN cell body ( Figure 27F), while the endogenous α-syn is not detected by the human-specific antibody ( Figure 27E,G). Thus, overexpressed mutant Ala30Pro α-syn shows abnormal accumulation in the SN cell body and is a potential source of cellular stress. ...

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... then determined whether the presence of α-syn aggregates in SN DA fibers renders them more vulnerable to loss of Ret-mediated trophic support. I analyzed the DA fiber density in the striatum of 12-month-old control, TH-α-synA30P, DAT-Ret and DAT- Ret ;TH-α-synA30P animals using TH as a marker for DA axons ( Figure 27I-L). I found that DAT-Ret mice display a moderate loss of DA fibers innervating the striatum (30 %; n=5 mice/group; DAT-Ret vs. CTRL p<0,01, Student's t-test; Figure 27M), as previously mentioned (Figure 18). ...

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... analyzed the DA fiber density in the striatum of 12-month-old control, TH-α-synA30P, DAT-Ret and DAT- Ret ;TH-α-synA30P animals using TH as a marker for DA axons ( Figure 27I-L). I found that DAT-Ret mice display a moderate loss of DA fibers innervating the striatum (30 %; n=5 mice/group; DAT-Ret vs. CTRL p<0,01, Student's t-test; Figure 27M), as previously mentioned (Figure 18). Interestingly, the degree of DA fiber loss in 12-month-old DAT-Ret;TH-α-synA30P mice was not different from that of age- matched DAT-Ret mice (n=5 mice/group; p=NS, Student's t-test; Figure 27M), suggesting that DA axons deprived of Ret-mediated trophic support are not more vulnerable to the presence of misfolded alpha-synuclein. ...

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... found that DAT-Ret mice display a moderate loss of DA fibers innervating the striatum (30 %; n=5 mice/group; DAT-Ret vs. CTRL p<0,01, Student's t-test; Figure 27M), as previously mentioned (Figure 18). Interestingly, the degree of DA fiber loss in 12-month-old DAT-Ret;TH-α-synA30P mice was not different from that of age- matched DAT-Ret mice (n=5 mice/group; p=NS, Student's t-test; Figure 27M), suggesting that DA axons deprived of Ret-mediated trophic support are not more vulnerable to the presence of misfolded alpha-synuclein. ...

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... animals were chosen since the interaction between Ret and DJ-1 in controlling survival of SN cell bodies is maximal at this time point. I found surprisingly that 24-month-old TH-α-synA30P transgenic and DAT-Ret single mutant mice lose about 20 % of TH-positive DA neurons in the SN, relative to controls ( Figure 27N). This suggests that the presence of aggregated α- synA30P leads to the degeneration of a fraction of aging SN neurons. ...

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... DAT-Ret;TH-α-synA30P mice lose 36 % of TH-positive DA neurons in the SN, as compared to controls (n=5 mice/group; DAT-Ret;TH-α-synA30P vs. CTRL p<0.001 and DAT-Ret;TH-α-synA30P vs. DAT-Ret or TH-α-synA30P p<0.01, Student's t-test; Figure 27N). Comparison of neuronal loss in TH-α-synA30P, DAT-Ret single and DAT-Ret;TH-α-synA30P mutants suggests that SN neuron loss in DAT-Ret;TH-α- synA30P mice is additive and is not caused by a genetic interaction between trophic insufficiency (Ret loss) and increased cellular stress due to protein aggregation (α-syn overexpression); moreover, this result further suggests that distinct neurons are affected by loss of Ret function and α-syn aggregation. ...

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... Drosophila DJ-1B is ubiquitously expressed, DJ-1A appears to be enriched in certain tissues such as testes ( Menzies et al., 2005;Meulener et al., 2005). Using a DJ-1B specific antibody, I confirmed that endogenous DJ-1B is expressed in Drosophila heads at postnatal day 5; in addition, DJ-1B expression is lost in DJ-1A -/-;DJ-1B -/-flies ( Figure 28A). Similarly, Pontus Klein used RT-PCR to show that the DJ-1A transcript is present in fly heads (data not shown). ...

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... used the photoreceptor neuron specific glass multimer reporter (GMR) which allows transgene overexpression via the GAL4/UAS system in post-mitotic photoreceptor neurons in the developing retina, starting in late larval stages (Read et al., 2005). I found that increased levels of constitutively active Ret, Raf, ERK or increased levels of WT Akt1 did not modify endogenous DJ-1B levels ( Figure 28A). This suggests that the expression of DJ-1B is not modified following activation of Ret, Ras/ERK or Akt signaling. ...

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... confirmed that GMR-dRet MEN2B flies develop with retinal defects and then complemented this observation with a quantitative evaluation of the GMR-dRet MEN2B phenotype. The overall eye size in GMR-dRet MEN2B flies was decreased by 30 % compared to controls ( Figure 28B,C,E). I then sectioned the fly eyes and used a contrasting agent (toluidine blue) to highlight retinal organization. ...

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... then sectioned the fly eyes and used a contrasting agent (toluidine blue) to highlight retinal organization. I found, as previously reported, that numerous ommatidia are often fused together and display abnormal polarity and poorly patterned inter-ommatidial spaces ( Figure 28G). In addition, I found that the average ommatidium size was increased by 35% in GMR- dRet MEN2B mutants relative to controls (that carried the GMR-Gal4 construct; Figure 28G,I); a similar increase in photoreceptor cell size is seen in mutants overexpressing constitutively active Sevenless/Ras or PI3K/Akt components ( Rubin et al., 1997;Vanhaesebroeck et al., 1997). ...

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... found, as previously reported, that numerous ommatidia are often fused together and display abnormal polarity and poorly patterned inter-ommatidial spaces ( Figure 28G). In addition, I found that the average ommatidium size was increased by 35% in GMR- dRet MEN2B mutants relative to controls (that carried the GMR-Gal4 construct; Figure 28G,I); a similar increase in photoreceptor cell size is seen in mutants overexpressing constitutively active Sevenless/Ras or PI3K/Akt components ( Rubin et al., 1997;Vanhaesebroeck et al., 1997). To determine whether DJ-1 is a Ret interactor, I crossed GMR-dRet MEN2B flies with flies carrying DJ-1A and/or DJ-1B microdeletions that specifically inactivate DJ-1A or DJ-1B function (here named "knockout alleles") ( Meulener et al., 2005). ...

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... reducing DJ-1A/B activity lead to a strong suppression of GMR- dRet MEN2B phenotype ( Figure 28D,E), the resulting eyes having an improved overall organization. At the histological level, the ommatidial organization of GMR- dRet MEN2B flies heterozygous for DJ-1A (GMR-dRet MEN2B ;DJ-1A +/-) or DJ-1B (GMR-dRet MEN2B ;DJ-1B +/-), or lacking completely DJ-1A (GMR-dRet MEN2B ; DJ-1A -/-) was significantly restored and the size of ommatidial units was almost completely rescued ( Figure 28I). ...

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... reducing DJ-1A/B activity lead to a strong suppression of GMR- dRet MEN2B phenotype ( Figure 28D,E), the resulting eyes having an improved overall organization. At the histological level, the ommatidial organization of GMR- dRet MEN2B flies heterozygous for DJ-1A (GMR-dRet MEN2B ;DJ-1A +/-) or DJ-1B (GMR-dRet MEN2B ;DJ-1B +/-), or lacking completely DJ-1A (GMR-dRet MEN2B ; DJ-1A -/-) was significantly restored and the size of ommatidial units was almost completely rescued ( Figure 28I). Thus, DJ-1A/B activity is required for full manifestation of Ret MEN2B- induced eye phenotype. ...

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... next asked whether reducing DJ-1A/B activity ameliorates the retinal defects induced by another constitutively active Ret form (dRet MEN2A ). GMR-dRet MEN2A flies also display a rough eye phenotype and a reduction in total eye size ( Figure 28K,M). Remarkably, when DJ-1A/B function is reduced (GMR-dRet MEN2A ;DJ-1A +/-;DJ-1B -/- flies) the phenotype caused by Ret MEN2A overexpression is strongly suppressed ( Figure 28L,M). ...

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... MEN2A flies also display a rough eye phenotype and a reduction in total eye size ( Figure 28K,M). Remarkably, when DJ-1A/B function is reduced (GMR-dRet MEN2A ;DJ-1A +/-;DJ-1B -/- flies) the phenotype caused by Ret MEN2A overexpression is strongly suppressed ( Figure 28L,M). Therefore, DJ-1A/B activity is required for full manifestation of Ret MENA- induced eye phenotype. ...

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... then evaluated whether increasing DJ-1 activity is sufficient to further impair eye development in flies overexpressing constitutively active Ret MEN2 . For this, I used the moderate phenotype caused by Ret MEN2A overexpression in photoreceptor neurons using the GAL4/UAS system (GMR-Gal4/UAS-dRet MEN2A ; Figure 28O,Q). When DJ- 1A was co-expressed with Ret MEN2A in developing photoreceptor neurons (GMR- Gal4/UAS-dRet MEN2A /UAS-DJ1A WT ) the eye phenotype was further enhanced, and the resulting eyes became even smaller ( Figure 28P,Q 7917 ) are null alleles for DJ-1A or DJ- 1B, respectively. ...

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... this, I used the moderate phenotype caused by Ret MEN2A overexpression in photoreceptor neurons using the GAL4/UAS system (GMR-Gal4/UAS-dRet MEN2A ; Figure 28O,Q). When DJ- 1A was co-expressed with Ret MEN2A in developing photoreceptor neurons (GMR- Gal4/UAS-dRet MEN2A /UAS-DJ1A WT ) the eye phenotype was further enhanced, and the resulting eyes became even smaller ( Figure 28P,Q 7917 ) are null alleles for DJ-1A or DJ- 1B, respectively. I generated GMR-dRet MEN2B flies that carried these loss-of-function alleles and characterized their retinal organization and ultrastructure. ...

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... generated GMR-dRet MEN2B flies that carried these loss-of-function alleles and characterized their retinal organization and ultrastructure. I found remarkably, that the defects in eye and organization and size induced by overactive Ret MEN2B were strongly suppressed in these backgrounds of reduced DJ-1A/B activity; the resulting eyes displayed an improved retinal organization and restored eye size (Figure 29C-F,G). Similarly, the abnormal increase in ommatidia size and abnormal ommatidial fusion induced by overactive Ret MEN2B were significantly suppressed in a DJ-1A heterozygous background (GMR-dRet MEN2B / Df(2R) CX1 ; Figure 29H-K). ...

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... found remarkably, that the defects in eye and organization and size induced by overactive Ret MEN2B were strongly suppressed in these backgrounds of reduced DJ-1A/B activity; the resulting eyes displayed an improved retinal organization and restored eye size (Figure 29C-F,G). Similarly, the abnormal increase in ommatidia size and abnormal ommatidial fusion induced by overactive Ret MEN2B were significantly suppressed in a DJ-1A heterozygous background (GMR-dRet MEN2B / Df(2R) CX1 ; Figure 29H-K). Thus, the GMR-dRet MEN2B -mediated eye phenotype is partially suppressed in backgrounds of reduced DJ-1A/B activity. ...

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... of consitutively active Ras V12 in R7 photoreceptor neurons driven by the R7-neuron specific promoter sevenless (that is fused to the coding sequence for Ras V12 ; Sev-Ras V12 ) is sufficient to induce abnormal differentiation of additional cells into R7 photoreceptors; as a result, multiple R7 photoreceptors are induced in each ommatidium ( Karim et al., 1996). To test whether DJ-1A/B interacts genetically with activated Ras, I generated flies expressing Sev-Ras V12 in either a wild-type background or a background of reduced DJ-1A/B activity ( Figure 32). As expected, flies expressing Sev-Ras V12 in a wild-type background developed with rough eyes ( Figure 32B); histological examination of their retina revealed that the total number of photoreceptor neurons in each ommatidium increased to an average of 8,5 photoreceptors /ommatidium (P/O; Figure 32B',P). ...

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... test whether DJ-1A/B interacts genetically with activated Ras, I generated flies expressing Sev-Ras V12 in either a wild-type background or a background of reduced DJ-1A/B activity ( Figure 32). As expected, flies expressing Sev-Ras V12 in a wild-type background developed with rough eyes ( Figure 32B); histological examination of their retina revealed that the total number of photoreceptor neurons in each ommatidium increased to an average of 8,5 photoreceptors /ommatidium (P/O; Figure 32B',P). Sev-Ras V12 CTRL GMR/rl SEM Sev-Ras V12 Sev-Ras V12 Sev-Ras V12 DJ1A +/-DJ1B +/- +GMR/+DJ1A + GMR *** *** *** *** n.s. ...

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... test whether DJ-1A/B interacts genetically with activated Ras, I generated flies expressing Sev-Ras V12 in either a wild-type background or a background of reduced DJ-1A/B activity ( Figure 32). As expected, flies expressing Sev-Ras V12 in a wild-type background developed with rough eyes ( Figure 32B); histological examination of their retina revealed that the total number of photoreceptor neurons in each ommatidium increased to an average of 8,5 photoreceptors /ommatidium (P/O; Figure 32B',P). Sev-Ras V12 CTRL GMR/rl SEM Sev-Ras V12 Sev-Ras V12 Sev-Ras V12 DJ1A +/-DJ1B +/- +GMR/+DJ1A + GMR *** *** *** *** n.s. ...

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... reducing the DJ-1A/B levels (Sev-Ras V12 ;DJ-1A +/-;DJ-1B +/-), this phenotype was significantly rescued ( Figure 32C) the Sev-Ras V12 ;DJ-1A +/-;DJ-1B +/-flies displaying an improved retinal morphology while the number of P/O was restored ( Figure 32C',P). In addition, while in Sev-Ras V12 flies 67 % of ommatidia were abnormally fused with their neighbors, only 5 % abnormally fused ommatidia were detected in the rescued Sev-Ras V12 ;DJ-1A +/-;DJ-1B +/-flies ( Figure 32 C'and data not shown). ...

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... reducing the DJ-1A/B levels (Sev-Ras V12 ;DJ-1A +/-;DJ-1B +/-), this phenotype was significantly rescued ( Figure 32C) the Sev-Ras V12 ;DJ-1A +/-;DJ-1B +/-flies displaying an improved retinal morphology while the number of P/O was restored ( Figure 32C',P). In addition, while in Sev-Ras V12 flies 67 % of ommatidia were abnormally fused with their neighbors, only 5 % abnormally fused ommatidia were detected in the rescued Sev-Ras V12 ;DJ-1A +/-;DJ-1B +/-flies ( Figure 32 C'and data not shown). ...

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... reducing the DJ-1A/B levels (Sev-Ras V12 ;DJ-1A +/-;DJ-1B +/-), this phenotype was significantly rescued ( Figure 32C) the Sev-Ras V12 ;DJ-1A +/-;DJ-1B +/-flies displaying an improved retinal morphology while the number of P/O was restored ( Figure 32C',P). In addition, while in Sev-Ras V12 flies 67 % of ommatidia were abnormally fused with their neighbors, only 5 % abnormally fused ommatidia were detected in the rescued Sev-Ras V12 ;DJ-1A +/-;DJ-1B +/-flies ( Figure 32 C'and data not shown). Thus, DJ-1A/B activity is required for the full manifestation of the Ras V12 - induced eye phenotype. ...

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... test whether DJ-1 is sufficient to modulate the Ras V12 -mediated eye phenotype, I co-overexpressed Ras V12 in R7 neurons (Sev-Ras V12 ) and DJ-1A in all photoreceptor neurons (using the GMR driver). While Sev-Ras V12 and Sev-Ras V12 /GMR-Gal4 flies displayed the same moderate eye phenotype ( Figure 32D,D',P), Sev-Ras V12 /GMR/DJ- 1A eyes were severely affected and very rough ( Figure 32E). At the histological level, Sev-Ras V12 /GMR/DJ-1A eyes displayed a strong increase in the number of P/O (11 on average) compared to Sev-RasV12 and Sev-Ras V12 /GMR-Gal4 retinas ( Figure 32E',P). ...

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... test whether DJ-1 is sufficient to modulate the Ras V12 -mediated eye phenotype, I co-overexpressed Ras V12 in R7 neurons (Sev-Ras V12 ) and DJ-1A in all photoreceptor neurons (using the GMR driver). While Sev-Ras V12 and Sev-Ras V12 /GMR-Gal4 flies displayed the same moderate eye phenotype ( Figure 32D,D',P), Sev-Ras V12 /GMR/DJ- 1A eyes were severely affected and very rough ( Figure 32E). At the histological level, Sev-Ras V12 /GMR/DJ-1A eyes displayed a strong increase in the number of P/O (11 on average) compared to Sev-RasV12 and Sev-Ras V12 /GMR-Gal4 retinas ( Figure 32E',P). ...

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... Sev-Ras V12 and Sev-Ras V12 /GMR-Gal4 flies displayed the same moderate eye phenotype ( Figure 32D,D',P), Sev-Ras V12 /GMR/DJ- 1A eyes were severely affected and very rough ( Figure 32E). At the histological level, Sev-Ras V12 /GMR/DJ-1A eyes displayed a strong increase in the number of P/O (11 on average) compared to Sev-RasV12 and Sev-Ras V12 /GMR-Gal4 retinas ( Figure 32E',P). Thus, DJ-1 activity appears sufficient to modulate the Ras V12 -mediated eye development. ...

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... overexpressed rl SEM in all photoreceptor neurons using the GMR driver. The resulting GMR/rl SEM eyes were rough, slightly reduced in size and relatively disorganized compared to control GMR-Gal4 eyes ( Figure 32L). When analyzing the retinal ultrastructure, I found an increase in the number of P/O (9,5 P/O on average; Figure 32Q). ...

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... resulting GMR/rl SEM eyes were rough, slightly reduced in size and relatively disorganized compared to control GMR-Gal4 eyes ( Figure 32L). When analyzing the retinal ultrastructure, I found an increase in the number of P/O (9,5 P/O on average; Figure 32Q). I then generated flies that co-overexpress rl SEM and DJ-1A (GMR/rl SEM /DJ-1A) or DJ-1B (GMR/rl SEM /DJ-1B); I also generated flies overexpressing rl SEM in a DJ-1B heterozygous knockout background (GMR/rl SEM ;DJ- 1B +/-). ...

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... then generated flies that co-overexpress rl SEM and DJ-1A (GMR/rl SEM /DJ-1A) or DJ-1B (GMR/rl SEM /DJ-1B); I also generated flies overexpressing rl SEM in a DJ-1B heterozygous knockout background (GMR/rl SEM ;DJ- 1B +/-). The rough eye phenotype induced by rl SEM overexpression was not further enhanced after DJ-1A/B overexpression ( Figure 32M,N) nor did a decrease in DJ-1B activity exert a suppressing effect on the rl SEM -mediated phenotype ( Figure 32O); at the histological level, the increase in the number of P/O caused by rl SEM overexpression was not further modulated after modifying the levels of DJ-1A/B ( Figure 32M'-O',Q). Because DJ-1A/B activity failed to further modulate the constitutively active rolled signaling, DJ-1A/B function either upstream of Rolled/ERK or in parallel to the Rolled/ERK signaling pathway to control cell differentiation and proliferation in the retina. ...

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... then generated flies that co-overexpress rl SEM and DJ-1A (GMR/rl SEM /DJ-1A) or DJ-1B (GMR/rl SEM /DJ-1B); I also generated flies overexpressing rl SEM in a DJ-1B heterozygous knockout background (GMR/rl SEM ;DJ- 1B +/-). The rough eye phenotype induced by rl SEM overexpression was not further enhanced after DJ-1A/B overexpression ( Figure 32M,N) nor did a decrease in DJ-1B activity exert a suppressing effect on the rl SEM -mediated phenotype ( Figure 32O); at the histological level, the increase in the number of P/O caused by rl SEM overexpression was not further modulated after modifying the levels of DJ-1A/B ( Figure 32M'-O',Q). Because DJ-1A/B activity failed to further modulate the constitutively active rolled signaling, DJ-1A/B function either upstream of Rolled/ERK or in parallel to the Rolled/ERK signaling pathway to control cell differentiation and proliferation in the retina. ...

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... then generated flies that co-overexpress rl SEM and DJ-1A (GMR/rl SEM /DJ-1A) or DJ-1B (GMR/rl SEM /DJ-1B); I also generated flies overexpressing rl SEM in a DJ-1B heterozygous knockout background (GMR/rl SEM ;DJ- 1B +/-). The rough eye phenotype induced by rl SEM overexpression was not further enhanced after DJ-1A/B overexpression ( Figure 32M,N) nor did a decrease in DJ-1B activity exert a suppressing effect on the rl SEM -mediated phenotype ( Figure 32O); at the histological level, the increase in the number of P/O caused by rl SEM overexpression was not further modulated after modifying the levels of DJ-1A/B ( Figure 32M'-O',Q). Because DJ-1A/B activity failed to further modulate the constitutively active rolled signaling, DJ-1A/B function either upstream of Rolled/ERK or in parallel to the Rolled/ERK signaling pathway to control cell differentiation and proliferation in the retina. ...

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... BDNF was found to promote survival of cultured embryonic DA neurons (Hyman et al., 1991). Our finding that Ret and TrkB signaling are dispensable for the initial maintenance of the DA system (Figures 18 and 23; see also Kramer et al., 2007) was unexpected and suggests several possibilities. First, the removal of both Ret and TrkB function was defined by the activity of the Nestin or DAT promoters (which induced the expression of the Cre recombinase); the Nestin promoter induces Cre expression after E10.5 ( Kramer et al., 2006;Tronche et al., 1999) while the DAT promoter is activated at E15 ( Zhuang et al., 2005). ...

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... contrast to TrkB, we found that Ret signaling is crucial for long-term maintenance of SN axons and cell bodies (Figures 17, 18, 23, 24). We observed that aging mice lacking Ret signaling in DA neurons display a marked loss of DA axons innervating the striatum (up to 60 % in 24-month-old mice), while the loss of SN cell bodies was relatively moderate (about 30 % in 24-month-old mice). ...

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... mice display a normal complement of SN neurons at 3 months ( Figure 23 and Kramer et al., 2007) while degeneration in the SN starts during aging (12-24 months; Figures 18 and 23). Why do aging, but not adult DA neurons require Ret- mediated signaling for survival? ...

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... mice display a normal complement of SN neurons at 3 months ( Figure 23 and Kramer et al., 2007) while degeneration in the SN starts during aging (12-24 months; Figures 18 and 23). Why do aging, but not adult DA neurons require Ret- mediated signaling for survival? ...

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... these animals developed normally and showed no overt behavioral alterations, we found surprisingly that combined loss of Ret and DJ-1 leads to enhanced loss of midbrain DA neurons as compared to mice lacking Ret alone. About 40 % SN cell bodies were lost in 24-month-old Ret/DJ-1 mice, compared to only 25 % in mice lacking Ret function ( Figure 23). The interaction between Ret and DJ-1 is restricted to SN neurons (the VTA neurons are not affected) and is age-dependent, as adult (3-month-old) DAT-Ret/DJ-1 mice do not display DA neurodegeneration. ...

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... DAT-Ret mice the degree of fiber loss (50%) exceeds the degree of SN cell body loss (25 %); this suggests that a fraction of aging SN neurons deprived of Ret- mediated signaling might have lost their target innervation. When analyzing the density of DA fibers in DAT-Ret/DJ-1 mice, we found interestingly that it matches exactly the degree of fiber loss in DAT-Ret mice (50 % in both groups, Figure 25). The simplest explanation is that additional DJ-1 removal caused degeneration of Ret- deficient SN cell bodies that lost their target innervation (the target deprived pool), while Ret-deficient SN cell bodies with functional connections were largely unaffected ( Figure 35). ...

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... detected an increased density of reactive astrocytes in DAT-Ret mice, specifically during aging (24 months) and in the striatum (Figures 20, 26). Two cellular entities are altered in the striatum of 24-month-old DAT-Ret mice as compared to age- matched controls: the DA terminals projecting from the SN are markedly depleted (Figures 18 and 25) and the post-synaptic medium spiny neurons (MSNs) downregulated the neuronal marker NeuN and the post-synaptic DA marker DARPP- 32, thereby suggesting cellular dysfunction (Figure 19). ...

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... detected an increased density of reactive astrocytes in DAT-Ret mice, specifically during aging (24 months) and in the striatum (Figures 20, 26). Two cellular entities are altered in the striatum of 24-month-old DAT-Ret mice as compared to age- matched controls: the DA terminals projecting from the SN are markedly depleted (Figures 18 and 25) and the post-synaptic medium spiny neurons (MSNs) downregulated the neuronal marker NeuN and the post-synaptic DA marker DARPP- 32, thereby suggesting cellular dysfunction (Figure 19). This suggests that loss of pre- synaptic DA input leads to post-synaptic alterations in dopaminoceptive cells. ...

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... reactive astrocytes might not be directly involved in the DA fiber degeneration process, but are recruited after the onset of nigrostriatal degeneration. Moreover, since no increased density of astrocytes was detected in the SN (where DA neurodegeneration was ongoing; Figure 20) it appears that dysfunctional DA axons and/or MSN postsynaptic neurons, rather than SN cell bodies, generate recruiting signals for reactive astrocytes. ...

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... contrast to the astrogliosis in the striatum of aging DAT-Ret mice, the microglial response we detected was restricted to the substantia nigra ( Figure 21). Similar to astrocytes, microglial cells were also recruited after the degeneration occurred (at 24 but not 12 months). ...

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... more interesting is our finding that loss of Ret and DJ-1 signaling affects a subpopulation of SN neurons that project exclusively to the striatum. We found a specific requirement for Ret and DJ-1 activity in the GIRK2-positive subpopulation, while the other Calbindin-positive subpopulation did not require Ret/DJ-1signaling for survival (Figure 24). Interestingly, Calbindin-positive neurons in the SN were found to be specifically spared in PD ( Yamada et al., 1990) and in mice treated with the PD-causing toxin MPTP ( Liang et al., 1996). ...

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... of Ala30Pro human α-syn in DA neurons was shown to be insufficient to trigger overt SN neurodegeneration in these mice, up to 12 months of age, despite the presence of aggregated alpha-synuclein in the SN cell body (RathkeHartlieb et al., 2001). When aging DAT-Ret;TH-α-synA30P mice were generated and aged, we found surprisingly that they display an accelerated loss of SN cell bodies, relative to DAT-Ret mice (Figure 27). We also found, however that overexpression of mutant α-syn alone leads to a mild SN neurodegeneration (20 % loss of cell bodies) in 24-month-old animals. ...

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... the increased DA neurodegeneration in DAT-Ret;TH-α- synA30P mice is due to an additive, but not synergistic effect of Ret and DJ-1 deletion on SN neuron survival. We reached a similar conclusion when analyzing the maintenance of DA fibers in DAT-Ret;TH-α-synA30P mice ( Figure 27); the degeneration of DA fibers caused by loss of Ret was not further enhanced when mutant α-syn was overexpressed in SN neurons. These results suggest that Ret- deprived DA neurons are not more sensitive to the cellular stress caused by misfolded α-syn. ...

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... constitutively associated versions of Ret (Ret MEN2A and Ret MEN2B ) cause multiple endocrine neoplasia in humans, a cancer of neural crest origin ( Read et al., 2005). Targeted expression of these active Ret versions in the developing photoreceptor neurons of the fly disrupts the normal development of photoreceptors by promoting excessive cell proliferation, abnormal differentiation and by increasing their size ( Figures 28, 29); these effects are followed by a secondary apoptotic wave that leads to adult flies of reduced size and rough eye morphology ( Read et al., 2005). When DJ- 1A/B function is decreased, these retinal defects are significantly rescued and the resulting rescued eyes have a completely restored size. ...

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... DJ- 1A/B function is decreased, these retinal defects are significantly rescued and the resulting rescued eyes have a completely restored size. Conversely, DJ-1A overexpression is sufficient to enhance the Ret MEN2A phenotype (Figures 28, 29). Therefore, DJ-1A/B represent novel interactors of Ret signaling in Drosophila. ...

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... emerging evidence suggests lipid phosphatase-independent roles of PTEN ( Gao et al., 2000) the best studied being a protein-phosphatase-dependent inhibition of Ras/MAPK signaling ( Kerr et al., 2006;Nayeem et al., 2007), modulation of JNK signaling ( Vivanco et al., 2007) and several nuclear functions that involve protein- protein interactions, to control cell cycle progression maintenance of genomic stability or apoptosis (Yin and Shen, 2008). Drosophila (Fortini et al., 1992) and constitutively active Ras/ERK signaling severely impairs eye development ( Figure 32). We found surprisingly that DJ-1 modulated the eye phenotype of constitutively active Ras V12 (Figure 32), suggesting that DJ-1 functions downstream of Ras activation or in parallel to Ras-mediated signaling to mediate photoreceptor neuron development. ...

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... (Fortini et al., 1992) and constitutively active Ras/ERK signaling severely impairs eye development ( Figure 32). We found surprisingly that DJ-1 modulated the eye phenotype of constitutively active Ras V12 (Figure 32), suggesting that DJ-1 functions downstream of Ras activation or in parallel to Ras-mediated signaling to mediate photoreceptor neuron development. To map the interaction site between DJ-1 and Ras/MAPK signaling, we used a constitutively active version of ERK/rolled, which also induces defects in eye development when targeted to developing photoreceptor neurons. ...

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... map the interaction site between DJ-1 and Ras/MAPK signaling, we used a constitutively active version of ERK/rolled, which also induces defects in eye development when targeted to developing photoreceptor neurons. The abnormal proliferation and eye roughness induced by constitutively active rolled were not modulated by DJ-1A/B (Figure 32), suggesting that DJ-1 functions either upstream or in parallel to ERK activation. Preliminary evidence suggests that DJ-1 does not modulate the phenotype of constitutively active Raf, further restricting the interaction site between DJ-1 and the Ras/Raf/MEK/ERK pathway to Ras/Raf. ...