Neurogranin as Parkinson's and Alzheimer's disease stage markers
Recent findings of morphological and functional changes in Parkinson's disease brains have shown altered synapse formation, but their role in cognitive decline is still an area under exploration. Here we measured the concentration of three key synaptic proteins, Rab3A, SNAP25 and neurogranin by enzyme-linked immunosorbent assay, in cerebrospinal fluid from a total of 139 participants (87 controls and 52 Parkinson's disease patients out of which 30 were drug-naïve) and explored their associations with motor and cognitive symptoms. Associations with motor disease stage (assessed by Hoehn and Yahr scale) and cognitive performance (assessed by the Montreal Cognitive Assessment scores) were explored. An overall increase in the concentration of SNAP25 was found in Parkinson's disease patients (p?=?0.032). Increased neurogranin levels were found in the drug naïve patients subgroup (p?=?0.023). Significant associations were observed between increased concentration of neurogranin and cognitive impairment in total Parkinson's disease group (p?=?0.017), as well as in the drug naïve (p?=?0.021) and with motor disease stage (p?=?0.041). There were no significant disease-driven changes observed in the concentration of Rab3a. Concentrations SNAP25 and neurogranin were increased in cerebrospinal fluid of Parkinson's disease patients in a disease specific manner and related to cognitive and motor symptom severity. Future longitudinal studies should explore whether cerebrospinal fluid synaptic proteins can predict cognitive decline in Parkinson's disease.
INTRODUCTION: Biomarkers monitoring synaptic degeneration/loss would be valuable for Alzheimer's disease (AD) diagnosis. Postsynaptic protein neurogranin may be a promising cerebrospinal fluid (CSF) biomarker but has not yet been evaluated as a plasma biomarker.
METHODS: Using an in-house designed prototype enzyme-linked immunosorbent assay (ELISA) targeting neurogranin C-terminally, we studied neurogranin in paired CSF/plasma samples of controls (n = 29) versus patients experiencing MCI, or dementia, due to AD (in total n = 59).
RESULTS: CSF neurogranin was increased in AD and positively correlated with CSF tau, whereas there was a negative relationship between CSF neurogranin (and tau) and CSF Aβ1-42/Aβ1-40. No differences were detected in plasma neurogranin between controls and AD. Also, there was no correlation between CSF and plasma neurogranin, excluding confounding effects of the latter.
DISCUSSION: This study strengthens the potential of neurogranin as an AD CSF biomarker, which now needs validation in larger studies. As tools, straightforward immunoassays can be used, as demonstrated by the described ELISA.
The striatum, a major component of the brain basal nuclei, is central for planning and executing voluntary movements and undergoes lesions in neurodegenerative disorders such as Huntington disease. To perform highly integrated tasks, the striatum relies on a complex network of communication within and between brain regions with a key role devoted to secreted molecules. To characterize the rat striatum secretome, we combined in vivo microdialysis together with proteomics analysis of trypsin digests and peptidomics studies of native fragments. This versatile approach, carried out using different microdialysis probes and mass spectrometer devices, allowed evidencing with high confidence the expression of 88 proteins and 100 processed peptides. Their secretory pathways were predicted by in silico analysis. Whereas high molecular weight proteins were mainly secreted by the classical mode (94%), low molecular weight proteins equally used classical and non-classical modes (53 and 47%, respectively). In addition, our results suggested alternative secretion mechanisms not predicted by bioinformatics tools. Based on spectrum counting, we performed a relative quantification of secreted proteins and peptides in both basal and neuronal depolarization conditions. This allowed detecting a series of neuropeptide precursors and a 6-fold increase for neurosecretory protein VGF and proenkephalin (PENK) levels. A focused investigation and a long peptide experiment led to the identification of new secreted non-opioid PENK peptides, referred to as PENK 114-133, PENK 239-260, and PENK 143-185. Moreover we showed that injecting synthetic PENK 114-133 and PENK 239-260 into the striatum robustly increased glutamate release in this region. Thus, the combination of microdialysis and versatile proteomics methods shed new light on the secreted protein repertoire and evidenced novel neuropeptide transmitters.
A 13-kilobase pair genomic DNA encoding a 78-amino acid brain-specific calmodulin-binding protein kinase C (PKC) substrate, neurogranin (Ng/RC3; also known as RC3 or p17), has been sequenced. The Ng/RC3 gene is composed of four exons and three introns, with the protein-coding region located in the first and second exons. This gene was found to have multiple transcriptional start sites clustered within 20 base pairs (bp); it lacks the TATA, GC, and CCAAT boxes in the proximal upstream region of the start sites. The promoter activity was characterized by transfection of 293 cells with nested deletion mutants of the 5'-flanking region fused to the luciferase reporter gene. A minimal construct containing bp +11 to +256 was nearly as active as that covering bp -1508 to +256, whereas a shorter one covering bp +40 to +256 had a greatly reduced activity. Between bp +11 and +40 lies a 12-nucleotide sequence (CCCCGCCCACCC) containing overlapping binding sites for AP2 (CCGCCCACCC) and SP1 (CCCGCC); this region may be important for conferring the basal transcriptional activity of the Ng/RC3 gene. The expression of a Ng/RC3-luciferase fusion construct (-1508/+256) in transfected 293 cells was stimulated by phorbol 12-myristate 13-acetate (PMA), but not by cAMP, arachidonic acid, vitamin D, retinoic acid, or thyroxines T3 and T4. PMA caused a 2-4-fold stimulation of all the reporter gene constructs ranging from +11/+256 to -1508/+256. The stimulatory effects of PMA could be magnified by cotransfection with both Ca(2+)-dependent and -independent phorbol ester-binding PKC-alpha, -beta I, -beta II, -gamma, -delta, and -epsilon cDNAs, but not by non-phorbol ester-binding PKC-zeta cDNA. The Ng/RC3 and PKC-gamma genes have a similar expression pattern in the brain during development. These two genes share at least four conserved sequence segments 1.5 kilobase pair upstream from their transcriptional start sites and a gross similarity in that they possess several AT-rich segments within bp -550 to -950. A near homogeneous 20-kDa DNA-binding protein purified from rat brain was able to bind to these AT-rich regions of both Ng/RC3 and PKC-gamma genes with footprints containing ATTA, ATAA, and AATA sequences.