Immunohistochemical analysis reveals species differences in the distribution of DJ-1 protein in human versus rat brain. In the human brain (A-D), DJ-1 immunostaining was relatively evenly distributed across all cortical layers (A). At higher magnification, DJ-1 immunoreactivity was observed in glia in human cortical gray matter (B) and white matter (C). Very few neurons were labeled, but occasionally weak DJ-1 label was seen in human cortical neurons (D). In the rat brain (E-N), intense DJ-1 immunostaining was observed in neurons throughout the frontal cortex (E), with particularly strong label occurring in layer V. Neurons were also strongly labeled in layer II (F). The hallmark apical dendrite of some pyramidal neurons was visibly stained (G, arrowheads), and DJ-1 was also infrequently seen in cortical glia (H). DJ-1 immunoreactivity was present in neurons throughout the rat striatum (I-K) and substantia nigra (L-N). At higher magnification, DJ-1 label was observed in the nucleus, cytoplasm, and processes of neurons with layer V of the frontal cortex (G, arrowheads), the striatum (J and K, arrowheads), substantia nigra pars compacta (M, arrowheads), and substantia nigra pars reticulata (N, arrowheads). SNpc, substantia nigra pars compacta; SNpr, substantia nigra pars reticulata. Scale bar = 20 μm in B-D, G, H, J, K, M, and N; 50 μm in F; 100 μm in A, E, I, and L.

Comparison of DJ-1 and GFAP immunostaining in rat brain by dual immunofluorescence confocal microscopy. Rat brain sections were double stained with anti-DJ-1 and anti-GFAP antibodies. DJ-1-immunoreactive cells in the cortex, striatum, and substantia nigra were not labeled by GFAP antibodies (arrows). DJ-1 immunostaining was absent from most GFAP-immunoreactive astrocytes (open arrows), except occasional astrocytes in the white matter (arrowheads). Superimposed images (overlay) show the distributions of these two proteins and their overlap. Scale bar = 50 μm.

Immunohistochemical analysis reveals DJ-1 expression in human nigral and striatal neurons. DJ-1 protein is expressed in neurons throughout the human substantia nigra (A). In the substantia nigra pars compacta (B) and parts reticulata (C) DJ-1 immunostaining was observed in the perikarya and neurites. At higher magnification, DJ-1 immunoreactivity can be seen in neuromelanin-positive (D, arrowhead) as well as in neuromelanin-negative neurons (D). In human striatum (E and F), DJ-1 protein appears to be moderately expressed in most medium spiny neurons. Scale bar = 25 μm in D and F; 50 μm in E; 100 μm in B and C; 250 μm in A.

Electron micrographs illustrating the localization of DJ-1 in the neuropil of macaque monkey striatum. DJ-1 label was observed in all types of neuronal profiles, including axons (A and C, arrowheads), dendrites (B and C, arrows), presynaptic terminals (D, arrowhead), dendritic spines (E, arrowhead), and glia (F, arrowheads). Glial profiles were irregularly shaped and often observed in close proximity to synapses. Scale bar = 0.5 μm.

Characterization of anti-DJ-1 antibodies by Western blot and immunohistochemical analysis. (A) Equal amounts of homogenates (50 μg protein per lane) from wild-type (WT) mouse brain, DJ-1 knockout (KO) mouse brain, rat brain, macaque cortex, and human cortex were separated by SDS-PAGE and analyzed by immunoblotting (IB) with anti-DJ-1 antibodies P7C, P7F, or SA100 (KAM-SA100, Stressgen) and anti-α-synuclein antibodies as indicated. Immunoprecipitated HA-tagged human DJ-1 protein was used as a positive control. (B) Homogenates of macaque cortex (50 μg protein per lane) were resolved by SDS-PAGE and analyzed by immunoblotting. Anti-DJ-1 antibodies were preabsorbed for 2 h with 50 μg of synthetic human DJ-1 carboxyl-terminal peptide (DJ-1 C-Peptide, amino acids 171−189) or purified recombinant human DJ-1 (DJ-1 protein) as indicated, prior to immunoblotting. (C) Immunohistochemical analysis of DJ-1 protein distribution in human cortical tissues using P7C, P7F, or SA100 demonstrates similar labeling of astrocytes. Scale bar = 50 μm.

DJ-1 expression in dopaminergic neurons as well as in other neurons in rat brain. Rat brain sections were double stained with anti-DJ-1 antibody and antibodies against NeuN or tyrosine hydroxylase (TH) as indicated. DJ-1 label was observed in NeuN-immunoreactive neurons in the cortex, striatum, and substantia nigra (A, arrowheads). In addition, DJ-1 antibodies stained nigral, tyrosine hydroxylase-immunoreactive neurons (B, arrowheads). There are also many NeuN-immunoreactive neurons that were not labeled by DJ-1 antibodies (A, open arrowheads). DJ-1 immunostaining was occasionally observed in white matter glia, which were unstained by NeuN antibodies (A, arrows). Superimposed images (overlay) demonstrate the overlap in the expression patterns of DJ-1 with NeuN or TH. Scale bar = 50 μm.

DJ-1 is expressed in macaque nigral and striatal neurons. DJ-1 immunostaining was distributed across all cortical layers within macaque frontal cortex (A). At higher magnification, DJ-1 immunoreactivity was found in glial cells in the cortical gray matter (B) and white matter (C), as well as in some pyramidal neurons (D). The macaque striatum (E) displays intense DJ-1 immunostaining, with immunoreactivity in medium spiny neurons (F), large interneurons (E, arrow), and some glial cells (G). DJ-1 immunostaining is also found in neurons in the substantia nigra pars compacta and their processes (H) as well as in neuronal perikarya in the ventral tegmental area (I). Scale bar = 20 μm in B-D, F, and G; 50 μm in E, H, and I; 100 μm in A.

Electron micrographs illustrating the localization of DJ-1 in the neuropil of macaque monkey prefrontal cortex. DJ-1 label was primarily observed in glia (A and B, arrowheads) and dendrites (C and D, arrows). Glial profiles were irregularly shaped and often wrapped around asymmetric synaptic contacts. Some labeled dendritic profiles received asymmetric synaptic contacts (D, arrowheads). Scale bar = 0.5 μm.

Quantitative analysis reveals a selective enrichment of DJ-1 protein in striatal axons, presynaptic terminals, and dendritic spines compared to the cortical DJ-1 distribution. Histograms show the relative distribution of DJ-1-immunreactive elements in the striatum (a total of 615 DJ-1-labeled profiles collected from three monkeys) and in the prefrontal cortex (a total of 584 labeled profiles collected from three monkeys). Chi-square analysis revealed a significant difference between the distribution of DJ-1 in the striatum and prefrontal cortex. Asterisk indicates statistically significant (p < 0.0001) difference in the number of DJ-1 immunoreactive profiles in the striatum versus that in the prefrontal cortex.