Upregulation and axonal transport of synaptotagmin‐IV in the direct‐pathway medium spiny neurons in hemi‐parkinsonian rats induced by dopamine D1 receptor stimulation

Abstract

Synaptotagmin-IV (Syt-IV) may function as a regulator of Ca(2+) -dependent synaptic transmission. In the hemi-parkinsonian rats with unilateral lesions of dopaminergic nigrostriatal neurons Syt-IV and substance-P (SP) mRNAs could be upregulated within the dopaminergically hypersensitive striatum of the lesioned brain hemisphere via the stimulation of striatal dopamine D1 (D1-R), but not D2 receptors. The hypersensitive D1-R-mediated transmission may be the culprit for the undesired expression of levodopa-induced dyskinesia, implying the involvement of Syt-IV and SP in the process. First, striatal cellular phenotypes expressing Syt-IV were determined. It was found to be expressed in all striatal neurons and a small population of astrocytes. Then it was examined, if the D1-R-mediated upregulation of Syt-IV mRNA may result in the upregulation of the translated protein. It was found that, after acute stimulation with a selective D1 agonist, (±)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF-82958), Syt-IV was elevated within the SP-expressing striatal neurons of the lesioned side. This was followed by the upregulation of Syt-IV, but not of its mRNA, within the ipsilateral target nuclei of the direct-pathway medium spiny neurons, indicating axonal transport of de novo synthesized protein to their SP-positive synaptic terminals. However, despite the striatal upregulation of SP and Syt-IV following a similar time-course, their subcellular co-localization within the axonal terminals was not found. It was therefore suggested that Syt-IV may regulate the hypersensitive striatal synaptic transmission, although via a SP-independent mechanism.

Are the Symptoms of Parkinsonism Cortical in Origin?

“…They play a role in the expression of sensitization to prolonged administration of L-DOPA, in the dopamine denervated animals that results in choreic, dystonic and ballistic movements called L-dopa-induced dyskinesia [31], [67].…”

Neuroinflammation-Induced Upregulation of Glial Cathepsin X Expression and Activity in vivo

“…The striatal and nigral sections (10 µm) of LPS treated rats (n = 5) were double-immunostained for the analysis of the cellular localization of cathepsin X using the following primary antibodies: goat polyclonal anti-cathepsin X antibody (1:75, R&D System), mouse monoclonal anti-TH antibody (1:500, Abcam) as a marker for dopaminergic neurons, mouse monoclonal anti-NeuN antibody (1:300, EMD Millipore) as a neuronal marker, mouse monoclonal anti-CD11b antibody (1: 175, Abcam) as microglia marker and mouse monoclonal anti-glial fibrillary acidic protein (GFAP) antibody (1:1000, Abcam) as astrocyte marker. Brain slices were fixed in cold methanol for 20 min and the immunofluorescence procedure was further performed as reported (Tratnjek et al, 2016). Briefly, brain slices were incubated in blocking solution containing 4% donkey serum (EMD Millipore) and 0.4% Triton X-100 in KPBS for 1 h at room temperature.…”

Therapeutic potential of inhibition of the neuroinflammation induced cathepsin X:in vivoevidence

“…The striatal and nigral sections (10 µm) of LPS treated rats were double-immunostained for the analysis of the cellular localization of cathepsin X using the following primary antibodies: goat polyclonal anticathepsin X antibody (1:75, R&D System), mouse monoclonal anti-TH antibody (1:500, Abcam) as marker for dopaminergic neurons, mouse monoclonal anti-NeuN antibody (1:300, EMD Millipore) as neuronal marker, mouse monoclonal anti-CD11b antibody (1: 175, Abcam) as microglia marker and mouse monoclonal anti-glial fibrillary acidic protein (GFAP) antibody (1:1000, Abcam) as astrocyte marker. Brain slices were fixed in cold methanol for 20 min and the immunofluorescence procedure was further performed as reported (Tratnjek et al, 2016). Briefly, brain slices were then incubated in blocking solution containing 4% donkey serum (EMD Millipore) and 0.4% Triton X-100 in KPBS for 1 h at room temperature.…”