Nigral-specific increase in ser31 phosphorylation compensates for tyrosine hydroxylase protein and nigrostriatal neuron loss: Implications for delaying parkinsonian signs.

Compensatory mechanisms that augment dopamine (DA) signaling are thought to mitigate onset of hypokinesia prior to major loss of tyrosine hydroxylase (TH) in striatum that occurs in Parkinson's disease. However, the identity of such mechanisms remains elusive. In the present study, the rat nigrostriatal pathway was unilaterally-lesioned with 6-hydroxydopamine (6-OHDA) to determine whether differences in DA content, TH protein, TH phosphorylation, or D1 receptor expression in striatum or substantia nigra (SN) aligned with hypokinesia onset and severity at two time points. In striatum, DA and TH loss reached its maximum (>90%) 7 days after lesion induction. However, in SN, no DA loss occurred, despite ∼60% TH loss. Hypokinesia was established at 21 days post-lesion and maintained at 28 days. At this time, DA loss was ∼60% in the SN, but still of lesser magnitude than TH loss. At day 7 and 28, ser31 TH phosphorylation increased only in SN, corresponding to less DA versus TH protein loss. In contrast, ser40 TH phosphorylation was unaffected in either region. Despite DA loss in both regions at day 28, D1 receptor expression increased only in lesioned SN. These results support the concept that augmented components of DA signaling in the SN, through increased ser31 TH phosphorylation and D1 receptor expression, contribute as compensatory mechanisms against progressive nigrostriatal neuron and TH protein loss, and may mitigate hypokinesia severity.

Differential expression of RET and GDNF family receptor, GFR-α1, between striatum and substantia nigra following nigrostriatal lesion: A case for diminished GDNF-signaling.

Although glial cell line-derived neurotrophic factor (GDNF) showed efficacy in preclinical and early clinical studies to alleviate parkinsonian signs in Parkinson's disease (PD), later trials did not meet primary endpoints, giving pause to consider further investigation. While GDNF dose and delivery methods may have contributed to diminished efficacy, one crucial aspect of these clinical studies is that GDNF treatment began ∼8 years after PD diagnosis; a time point representing several years after near 100% depletion of nigrostriatal dopamine markers in striatum and at least 50% in substantia nigra (SN), which represents a time point of initiating GDNF treatment later than reported in some preclinical studies. With nigrostriatal terminal loss exceeding 70% at PD diagnosis, we utilized hemiparkinsonian rats to determine if expression of GDNF family receptor, GFR-α1, and receptor tyrosine kinase, RET, differed between striatum and SN at 1 and 4 weeks following a 6-hydroxydopamine (6-OHDA) hemilesion. Whereas GDNF expression changed minimally, GFR-α1 expression decreased progressively in striatum and in tyrosine hydroxylase positive (TH+) cells in SN, correlating with reduced TH cell number. However, in nigral astrocytes, GFR-α1 expression increased. RET expression decreased maximally in striatum by 1 week, whereas in the SN, a transient bilateral increase occurred, returning to control levels by 4 weeks. Expression of brain-derived neurotrophic factor (BDNF) or its receptor, TrkB, were unchanged throughout lesion progression. Together, these results reveal that differential GFR-α1 and RET expression between the striatum and SN, and cell-specific differences in GFR-α1 expression in SN, occur during nigrostriatal neuron loss. Targeting loss of GDNF receptors thus appears critical to enhance GDNF therapeutic efficacy against nigrostriatal neuron loss. SIGNIFICANCE STATEMENT: Although preclinical evidence supports that GDNF provides neuroprotection and improves locomotor function in preclinical studies, there is uncertainty if it can alleviate motor impairment in Parkinson's disease patients. Using the established 6-OHDA hemiparkinsonian rat model, we determined whether expression of its cognate receptors, GFR-α1 and RET, were differentially affected between striatum and substantia nigra in a timeline study. In striatum, there was early and significant loss of RET, but a gradual, progressive loss of GFR-α1. In contrast, RET transiently increased in lesioned substantia nigra, but GFR-α1 progressively decreased only in nigrostriatal neurons and correlated with TH cell loss. Our results indicate that direct availability of GFR-α1 may be a critical element that determines GDNF efficacy following striatal delivery.

Differential expression of RET and GDNF family receptor, GFR-α1, between striatum and substantia nigra following nigrostriatal lesion: a case for diminished GDNF-signaling.

Although glial cell line-derived neurotrophic factor (GDNF) showed efficacy in preclinical and early clinical studies to alleviate parkinsonian signs in Parkinson's disease (PD), later trials did not meet primary endpoints, giving pause to consider further investigation. While GDNF dose and delivery methods may have contributed to diminished efficacy, one crucial aspect of these clinical studies is that GDNF treatment across all studies began ∼8 years after PD diagnosis; a time point representing several years after near 100% depletion of nigrostriatal dopamine markers in striatum and at least 50% in substantia nigra (SN), and is later than the timing of GDNF treatment in preclinical studies. With nigrostriatal terminal loss exceeding 70% at PD diagnosis, we utilized hemi-parkinsonian rats to determine if expression of GDNF family receptor, GFR-α1, and receptor tyrosine kinase, RET, differed between striatum and SN at 1 and 4 weeks following a 6-hydroxydopamine (6-OHDA) lesion. Whereas GDNF expression changed minimally, GFR-α1 expression decreased progressively in striatum and in tyrosine hydroxylase positive (TH+) cells in SN, correlating with reduced TH cell number. However, in nigral astrocytes, GFR-α1 expression increased. RET expression decreased maximally in striatum by 1 week, whereas in the SN, a transient bilateral increase occurred that returned to control levels by 4 weeks. Expression of brain-derived neurotrophic factor (BDNF) or its receptor, TrkB, were unchanged throughout lesion progression. Together, these results reveal that differential GFR-α1 and RET expression between the striatum and SN, and cell-specific differences in GFR-α1 expression in SN, occur during nigrostriatal neuron loss. Targeting loss of GDNF receptors appears critical to enhance GDNF therapeutic efficacy against nigrostriatal neuron loss. Significance Statement: Although preclinical evidence supports that GDNF provides neuroprotection and improves locomotor function in preclinical studies, clinical data supporting its efficacy to alleviate motor impairment in Parkinson's disease patients remains uncertain. Using the established 6-OHDA hemi-parkinsonian rat model, we determined whether expression of its cognate receptors, GFR-α1 and RET, were differentially affected between striatum and substantia nigra in a timeline study. In striatum, there was early and significant loss of RET, but a gradual, progressive loss of GFR-α1. In contrast, RET transiently increased in lesioned substantia nigra, but GFR-α1 progressively decreased only in nigrostriatal neurons and correlated with TH cell loss. Our results indicate that direct availability of GFR-α1 may be a critical element that determines GDNF efficacy following striatal delivery. Highlights: GDNF expression was minimally affected by nigrostriatal lesionGDNF family receptor, GFR-α1, progressively decreased in striatum and in TH neurons in SN.GFR-α1 expression decreased along with TH neurons as lesion progressedGFR-α1 increased bilaterally in GFAP+ cells suggesting an inherent response to offset TH neuron lossRET expression was severely reduced in striatum, whereas it increased in SN early after lesion induction.