The neuropathological substrate of dementia in patients with Parkinson's disease is still under debate, particularly in patients with insufficient alternate neuropathology for other degenerative dementias. In patients with pure Lewy body Parkinson's disease, previous post-mortem studies have shown that dopaminergic and cholinergic regulatory projection systems degenerate, but the exact pathways that may explain the development of dementia in patients with Parkinson's disease remain unclear. Studies in rodents suggest that both the mesocorticolimbic dopaminergic and septohippocampal cholinergic pathways may functionally interact to regulate certain aspects of cognition, however, whether such an interaction occurs in humans is still poorly understood. In this study, we performed stereological analyses of the A9 and A10 dopaminergic neurons and Ch1, Ch2 and Ch4 cholinergic neurons located in the basal forebrain, along with an assessment of α-synuclein pathology in these regions and in the hippocampus of six demented and five non-demented patients with Parkinson's disease and five age-matched control individuals with no signs of neurological disease. Moreover, we measured choline acetyltransferase activity in the hippocampus and frontal cortex of eight demented and eight non-demented patients with Parkinson's disease, as well as in the same areas of eight age-matched controls. All patients with Parkinson's disease exhibited a similar 80-85% loss of pigmented A9 dopaminergic neurons, whereas patients with Parkinson's disease dementia presented an additional loss in the lateral part of A10 dopaminergic neurons as well as Ch4 nucleus basalis neurons. In contrast, medial A10 dopaminergic neurons and Ch1 and Ch2 cholinergic septal neurons were largely spared. Despite variable Ch4 cell loss, cortical but not hippocampal cholinergic activity was consistently reduced in all patients with Parkinson's disease, suggesting significant dysfunction in cortical cholinergic pathways before frank neuronal degeneration. Patients with Parkinson's disease dementia were differentiated by a significant reduction in hippocampal cholinergic activity, by a significant loss of non-pigmented lateral A10 dopaminergic neurons and Ch4 cholinergic neurons (30 and 55% cell loss, respectively, compared with neuronal preservation in control subjects), and by an increase in the severity of α-synuclein pathology in the basal forebrain and hippocampus. Overall, these results point to increasing α-synuclein deposition and hippocampal dysfunction in a setting of more widespread degeneration of cortical dopaminergic and cholinergic pathways as contributing to the dementia occurring in patients with pure Parkinson's disease. Furthermore, our findings support the concept that α-synuclein deposition is associated with significant neuronal dysfunction in the absence of frank neuronal loss in Parkinson's disease.

Dynamic Nuclear Polarization (DNP) by the so-called 'dissolution' procedure 1 is rapidly gaining momentum as a novel method to enhance weak nuclear magnetic resonance (NMR) signals from molecular tracers, so that one can visualize their biodistribution and metabolism in ViVo. 2 The major limitation of the technique arises from the short lifetimes of hyperpolarized spin states in liquids. In particular, longitudinal relaxation times T 1 of protons in solutions of biomolecules are too short to allow for transport and in ViVo injection of hyperpolarized compounds. Most applications of the technique have therefore focused on 13 C NMR of 13 C-enriched tracers containing nonprotonated carbons with T 1 ( 13 C) ≈ 20-40 s. Choline (CH 3 ) 3 N + CH 2 CH 2 OH plays a key role in several critical biological processes, in particular in the synthesis and metabolism of phospholipids in cell membranes, and in cholinergic neurotransmission. Although the choline molecule does not contain any slowly relaxing carbons, it possesses a quaternary nitrogen with T 1 ( 15 N) > 120 s, which lends itself to hyperpolarization. 3 The conversion of choline to phosphocholine catalyzed by choline kinase has recently been monitored by 15 N NMR in Vitro, 3 employing hyperpolarization of 15 N spins. In ViVo measurements using this method may, however, be hampered by insufficient 15 N peak separation of choline metabolites (∼ 0.2 ppm for phosphocholine vs choline, i.e., only 6 Hz in a field B 0 ) 7 T) and by poor sensitivity of 15 N NMR. A sensitivity improvement by at least an order of magnitude would be required, e.g., to monitor phosphocholine accumulation in tumor cell cultures. 4 The above limitations may be overcome by transferring the hyperpolarization from 15 N to protons, as in recent heteronuclear 2D DNP-NMR experiments. 5 A similar concept was recently used for 13 C enhanced by PASADENA. 6 In this work, we show that one can transfer the long-lived 15 N hyperpolarization to remote methylene CH 2 O protons in choline across three bonds via 3 J( 15 N, 1 H), which significantly improves both the sensitivity and the spectral dispersion of choline metabolites. We also show that T 1 ( 15 N) in choline can be considerably increased by deuteration of the methyl groups.The conventional 1 H spectrum of 15 N-enriched choline is shown in Figure 1A. The peak at 3.19 ppm, which stems from the nine magnetically equivalent methyl protons, is commonly used for in ViVo quantification of choline-containing compounds, 7 whereas the multiplets due to the NCH 2 (3.50 ppm) and CH 2 O protons (4.05 ppm) 8 exhibit an AA′XX′ pattern. 9 The CH 2 O and methyl peaks have additional doublet structures due to 3 J( 15 N, 1 H) ≈ 3.7 Hz and 2 J( 15 N, 1 H) ≈ 0.8 Hz. (In nonenriched choline, one observes triplets due to 3 J( 14 N, 1 H) ) 2.7 Hz and 2 J( 14 N, 1 H) ) 0.6 Hz. 8,9 ) As shown in Figure 1B, the small n J( 15 N, 1 H) couplings in choline can be used to transfer hyperpolarization from 15 N to CH 2 O and methyl protons, using a reversed INEPT pulse sequence. 10,11...

Emerging research has re-emphasized the role of the cortical cholinergic system in the symptomology and progression of Alzheimer’s disease (AD). Basal forebrain (BF) cholinergic nuclei depend on target-derived NGF for survival during development and for the maintenance of a classical cholinergic phenotype during adulthood. In AD, BF cholinergic neurons lose their cholinergic phenotype and function, suggesting an impairment in NGF-mediated trophic support. We propose that alterations to the enzymatic pathway that controls the maturation of proNGF to mature NGF and the latter’s ulterior degradation underlie this pathological process. Indeed, the NGF metabolic pathway has been demonstrated to be impaired in AD and other amyloid pathologies, and pharmacological manipulation of NGF metabolism has consequences in vivo for both levels of proNGF/NGF and the phenotype of BF cholinergic neurons. The NGF pathway may also have potential as a biomarker of cognitive decline in AD, as its changes can predict future cognitive decline in patients with Down syndrome as they develop preclinical Alzheimer’s pathology. New evidence suggests that the cholinergic system, and by extension NGF, may have a greater role in the progression of AD than previously realized, as changes to the BF precede and predict changes to the entorhinal cortex, as anticholinergic drugs increase odds of developing AD, and as the use of donepezil can reduce rates of hippocampal and cortical thinning. These findings suggest that new, more sophisticated cholinergic therapies should be capable of preserving the basal forebrain thus having profound positive effects as treatments for AD.

BackgroundAlpha-synuclein (asyn) has been shown to play an important role in the neuropathology of Parkinson’s disease (PD). In the diseased brain, classic intraneuronal inclusions called Lewy bodies contain abnormal formations of asyn protein which is mostly phosphorylated at serine 129 (pS129 asyn). This suggests that post-translational modifications may play a role in the pathogenic process. To date, several uniplex assays have been developed in order to quantify asyn not only in the brain but also in cerebrospinal fluid and blood samples in order to correlate asyn levels to disease severity and progression. Notably, only four assays have been established to measure pS129 asyn specifically and none provide simultaneous readout of the total and pS129 species. Therefore, we developed a sensitive high-throughput duplex assay quantifying total and pS129 human asyn (h-asyn) in the same well hence improving accuracy as well as saving time, consumables and samples.ResultsUsing our newly established duplex assay we measured total and pS129 h-asyn in vitro showing that polo-like kinase 2 (PLK2) can phosphorylate asyn up to 41 % in HEK293 cells and in vivo the same kinase phosphorylated h-asyn up to 17 % in rat ventral midbrain neurons. Interestingly, no increase in phosphorylation was observed when PLK2 and h-asyn were co-expressed in rat striatal neurons. Furthermore, using this assay we investigated h-asyn levels in brain tissue samples from patients with PD as well as PD dementia and found significant differences in pS129 h-asyn levels not only between disease tissue and healthy control samples but also between the two distinct disease states especially in hippocampal tissue samples.ConclusionsThese results demonstrate that our duplex assay for simultaneous quantification is a useful tool to study h-asyn phosphorylation events in biospecimens and will be helpful in studies investigating the precise causative link between post-translational modification of h-asyn and PD pathology.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0125-0) contains supplementary material, which is available to authorized users.