Alterations in peptide levels in Parkinson's disease and incidental Lewy body disease

The levels of the neuropeptides Met- and Leu-enkephalin (MET-ENK, LEU-ENK), substance P and neurotensin were measured by a combined high performance liquid chromatography/radioimmunoassay (HPLC/RIA) method in postmortem samples of basal ganglia from Parkinson's disease patients, incidental Lewy body disease patients (presymptomatic Parkinson's disease) and matched controls. Dopamine (DA) levels were reduced in the caudate nucleus and putamen in Parkinson's disease, but unaltered in incidental Lewy body disease. The levels of MET-ENK were reduced in the caudate nucleus, putamen and substantia nigra in Parkinson's disease. Met-enkephalin levels were reduced in the caudate nucleus and in the putamen in incidental Lewy body disease. Leu-enkephalin levels were decreased in the putamen and were undetectable in the substantia nigra in Parkinson's disease. Leu-enkephalin levels were unchanged in incidental Lewy body disease, although there was a tendency to a reduction in putamen. Substance P levels were reduced in the putamen in Parkinson's disease. No significant changes in substance P content were observed in incidental Lewy body disease. Neurotensin levels were increased in the substantia nigra in Parkinson's disease. Neurotensin levels in incidental Lewy body disease were not altered significantly, but tended to parallel the changes in Parkinson's disease. The changes in basal ganglia peptide levels in incidental Lewy body disease generally followed a trend similar to those seen in Parkinson's disease, but were less marked. This suggests that they are an integral part of the pathology of the illness and not secondary to DA neuronal loss or a consequence of prolonged drug therapy.Peer Reviewe

Erythropoietin dependent regulation of the anti-apoptotic TMBIM family members FAIM2 and GRINA after murine cerebral ischemia

Cerebral ischemia is the most common type of stroke and one of the leading causes of disability and death worldwide. Ischemic stroke occurs due to an insufficient blood perfusion to the brain leading to rapid neuronal cell death in the infarct core. The adjacent penumbra region is meta-stable and heterogeneously susceptible to ischemia making it amenable to therapeutic interventions. The currently approved reperfusion modalities are systemic thrombolysis by recombinant tissue plasminogen activator (rtPA) and endo-vascular treatments (e.g. mechanical thrombectomy). Due to their time-dependency they are applicable for only 15% of stroke patients. Neuroprotective approaches have not been successful so far. Modulation of apoptosis and endogenous cytoprotective pathways may provide a promising therapeutic strategy after ischemic stroke. The evolutionary highly conserved and ubiquitously expressed transmembrane BAX Inhibitor-1 Motif-containing (TMBIM) protein family is attributed a significant role in the maintenance of intracellular calcium homeostasis and in the inhibition of apoptosis. TMBIM2/FAIM2 and TMBIM3/GRINA share a similar secondary structure and are found in membranes of the endoplasmic reticulum (ER) and/or in plasma membranes of the central nervous system (CNS). FAIM2 showed neuroprotective and regenerative effects in a murine transient middle cerebral artery occlusion (tMCAo) model and is regulated by erythropoietin (EPO). The role of GRINA in transient brain ischemia, its potential compensatory or synergistic effects with FAIM2 and its regulation by EPO treatment were not elucidated yet. To address this, tMCAo or Sham surgery for 30 minutes followed by a subsequent reperfusion time of 6 h and/or 72 h were performed in GRINA-deficient (Grina-/-), FAIM2-deficient (Faim2-/-), double-deficient (Grina-/-Faim2-/-) and wildtype littermate (WT) mice. EPO or saline were administered 0, 24 and 48 hours after tMCAo or Sham surgery. Neurological out- come was evaluated at various time points. Primary murine cortical neurons (pMCN) of all mouse strains were subjected to oxygen-glucose deprivation (OGD) after Grina and/or Faim2 gene transfection. High expression levels of both TMBIM family members were found in the murine brain. Grina-/- led to a similar increase in infarct volumes as Faim2-/- mice. The double-deficient mice revealed the highest neurological deficits and largest infarct sizes after stroke. EPO administration upregulated Grina and Faim2 mRNA levels in wildtype littermates. EPO significantly decreased infarct sizes and abrogated neurological impairments in WT mice only. While the absence of FAIM2 potentiated the activation of caspase 8, a strong activation of caspase 9 was observed in Grina-/- mice. Likewise, pMCN of FAIM2- and/or GRINA-deficient mice showed a pronounced impaired ischemia tolerance after OGD compared to WT. Over- expression of Grina and Faim2 in WT neurons and the reintroduction of both TMBIM genes alone and in combination in double-deficient neurons significantly decreased the OGD-induced cell death rates. Cerebral ischemia is known to cause the accumulation of misfolded proteins and loss of calcium homeostasis leading to endoplasmic reticulum (ER) stress. This activates an ER- located and cytoprotective pathway, the unfolded protein response (UPR). Since GRINA is mostly located at the ER membrane suppressing ER calcium release by inositol-1,4,5- trisphosphate receptors, we hypothesized that GRINA might play a crucial role in the UPR. Thus, we investigated the influence of GRINA and EPO on the post-ischemic UPR in a second study. We subjected Grina-/- and WT mice to 30 minutes of tMCAo or Sham surgery followed by 6 h or 72 h of reperfusion. We administered EPO or saline 0, 24 and 48 h after tMCAo/sham surgery. OGD and pharmacological stimulation of the UPR using Tunicamycin and Thapsigargin were carried out in primary murine cortical mixed cell cultures. Treatment with the PERK-inhibitor GSK-2606414, IRE1a-RNase-inhibitor STF-083010 and EPO was performed 1 h prior to 1 h, 2 h or 3 h of OGD. This study provided evidence that both the IREα and the PERK branch of the UPR are activated in the early reperfusion phase (6 h) after cerebral ischemia. Moreover, Grina-/- increased apoptosis and the activation of the corresponding PERK arm of the UPR after stroke compared to WT mice. EPO was able to enhance the post-ischemic activation of the protective IREα pathway and attenuated the pro-apoptotic PERK cascade. In addition to EPO, the PERK inhibitor GSK-2606414 also reduced post-ischemic cell death and regulated Grina transcription after ODG. In conclusion, both TMBIM family members play an important role in EPO-mediated neuro- protection after cerebral ischemia. FAIM2 appears to be involved in the extrinsic apoptosis pathway (caspase 8) and GRINA in the intrinsic apoptosis pathway (caspase 9). Further- more, GRINA seems to be implicated in the post-ischemic modulation of the UPR, particularly of the PERK arm. Besides the findings on the mechanism of EPO after cerebral ischemia, our studies also indicate a potential therapeutic relevance of PERK inhibitors after stroke

Dehydroepiandrosterone (DHEA) Serum Levels Indicate Cerebrospinal Fluid Levels of DHEA and Estradiol (E2) in Women at Term Pregnancy

Neuroactive steroids such as dehydroepiandrosterone (DHEA), estradiol (E2), and progesterone (P4) are associated with structural and functional changes in the central nervous system (CNS). Measurement of steroid levels in the CNS compartments is restricted in accessibility. Consequently, there is only limited human data on the distributional equilibrium for steroid levels between peripheral and central compartments. While some neuroactive steroids including DHEA and E2 have been reported to convey excitatory and proconvulsant properties, the opposite was demonstrated for P4. We aimed to elucidate the correlation between peripheral and central DHEA, E2, and P4 levels in women at term pregnancy. CSF and serum samples of 27 healthy pregnant women (22-39 years) at term pregnancy were collected simultaneously under combined spinal and epidural anesthesia and used for DHEA ELISA and E2, and P4 ECLIA. All three neuroactive steroids were detected at markedly lower levels in CSF compared to their corresponding serum concentrations (decrease, mean ± SD, 97.66 ± 0.83%). We found a strong correlation for DHEA between its serum and the corresponding CSF levels (r = 0.65, p = 0.003). Serum and CSF levels of E2 (r = 0.31, p = 0.12) appeared not to correlate in the investigated cohort. DHEA serum concentration correlated significantly with E2 (r = 0.58, p = 0.0016) in CSF. In addition, a strong correlation was found between DHEA and E2, both measured in CSF (r = 0.65, p = 0.0002). Peripheral DHEA levels might serve as an indicator for central nervous levels of the neuroactive steroids DHEA and E2 in pregnant women

CK1BP Reduces α-Synuclein Oligomerization and Aggregation Independent of Serine 129 Phosphorylation

The pathological accumulation of α-Synuclein (α-Syn) is the hallmark of neurodegenerative α-synucleinopathies, including Parkinsons’s disease (PD). In contrast to the mostly non-phosphorylated soluble α-Syn, aggregated α-Syn is usually phosphorylated at serine 129 (S129). Therefore, S129-phosphorylation is suspected to interfere with α-Syn aggregation. Among other kinases, protein kinase CK1 (CK1) is known to phosphorylate α-Syn at S129. We overexpressed CK1 binding protein (CK1BP) to inhibit CK1 kinase activity. Using Bimolecular Fluorescence Complementation (BiFC) in combination with biochemical methods, we monitored the S129 phosphorylation and oligomerization of α-Syn in HEK293T cells. We found that CK1BP reduced the overall protein levels of α-Syn. Moreover, CK1BP concomitantly reduced S129 phosphorylation, oligomerization and the amount of insoluble α-Syn. Analyzing different α-Syn variants including S129 mutations, we show that the effects of CK1BP on α-Syn accumulation were independent of S129 phosphorylation. Further analysis of an aggregating polyglutamine (polyQ) protein confirmed a phosphorylation-independent decrease in aggregation. Our results imply that the inhibition of CK1 activity by CK1BP might exert beneficial effects on NDDs in general. Accordingly, CK1BP represents a promising target for the rational design of therapeutic approaches to cease or at least delay the progression of α-synucleinopathies