05 Oct 2022

Scientists now have a slew of biomarkers, both fluid assays and PET ligands, that distinguish Alzheimer’s from other neurodegenerative diseases, such as frontotemporal dementia, Parkinson’s, and dementia with Lewy bodies (DLB). But in the great many cases where there is mixed pathology, the overlaps are messy and differential diagnosis often gets stuck. Could synaptic proteins ride to the rescue? At the 9th Kuopio Alzheimer’s Symposium held August 23-25 in this city in Eastern Finland, VAMP2 emerged as a candidate to distinguish pure DLB from DLB with evidence of concomitant AD pathology.

Olivia Belbin, IIB Sant Pau, Barcelona, Spain, reported that levels of this synaptic protein go down in the former, but up in the latter. “This suggests that different processes are at play in these diseases,” she said.

Henrik Zetterberg, University of Gothenburg, Sweden, was intrigued. “I know of no other marker that behaves like this,” he said, emphasizing that the findings need to be replicated. “If this is confirmed, then we really need to dig into cell-based mechanisms and other studies to find out what is going on with this marker,” he said.

VAMP2 stands for vesicle-associated membrane protein 2. It forms SNARE complexes with its fellow synaptic proteins SNAP-25 and Syntaxyn-1. These complexes help deliver synaptic vesicles to the presynapse and fuse them with the membrane there. Neurons can assemble and disassemble SNAREs thousands of times per minute. Scientists suspect SNAREs might prove to be sensors of change in synaptic health. Hence, they are studying whether SNARE levels in the parenchyma or CSF correlate with neurodegenerative pathologies in general, and with α-synucleinopathies in particular (see Cervantes-González and Belbin, 2022, for a review).

Older work from Nobel laureate Tom Sudhof’s lab indicates that α-synuclein promotes formation of SNARE complexes, a function that might be compromised by synuclein oligomers or aggregates (Burré et al., 2010). Last year, researchers in Italy reported that levels of VAMP2 in neuron-derived extracellular vesicles fell in people who had Parkinson’s disease (Agliardi et al., 2021). Those same scientists reported this year that people diagnosed with mixed dementia express more of the VAMP2 gene in their brains than do people who are cognitively normal or who have AD, and that these mixed dementia folk are more likely to have a 26 base pair deletion in an intergenic region of the gene (Costa et al., 2022). All told, interest in VAMP2 has recently blossomed.

Belbin was searching for markers that identify heterogeneities in neurodegenerative diseases. She noted that a majority of Alzheimer’s cases also have some other neuropathology, for example Lewy bodies, which comprise mostly α-synuclein aggregates. To find changes in VAMP2 in the CSF of people with AD and related disorders, Belbin developed a highly sensitive “homebrew” SIMOA immunoassay. It uses a rabbit monoclonal antibody from the company Cell Signaling Technology to capture the protein, plus a high-affinity mouse monoclonal developed by ADx NeuroSciences for detection. The assay's lower limit of quantification is 4.1 pg/mL, said Belbin.

Armed with this tool, Belbin measured CSF VAMP2 in samples from the Sant Pau Initiative for Neurodegeneration (Alcolea et al., 2019). Begun in 2011, SPIN enrolls healthy controls, people with subjective memory complaints and mild cognitive impairment, and people who have been diagnosed with AD, DLB, frontotemporal dementia, or Down’s syndrome.

In Kuopio, Belbin showed data from 63 healthy controls, 114 people with AD, 19 with pure DLB, and 28 with both DLB and AD pathology. On average, the latter had more VAMP2 in their CSF than did controls, but curiously, people with pure DLB had less VAMP2 than controls (see image below). In this way, the assay was able to distinguish people with pure DLB from those with DLB+AD with an AUC of 0.80—the AUC is a measure of sensitivity and specificity, with a value of 1.0 indicating a perfect assay.

Differential Diagnosis? These violin plots suggest that people with pure DLB have less VAMP2 in their CSF on average, than people with AD, or DLB+AD (left). In early AD, however, VAMP2 reaches a low point. [Courtesy of Olivia Belbin.]

Why the apparent difference between DLB and DLB+AD? Belbin thinks that in pure DLB, VAMP2 decreases in the CSF because it is sequestered by aggregates of α-synuclein. A recent mass spectrometry study spotted VAMP2 among a cadre of synaptic proteins that were embedded in Lewy bodies purified from people with DLB (McCormack et al., 2019). 

But why would VAMP2 increase in the CSF of AD patients? Belbin thinks this might reflect damage to synapses and/or neurodegeneration. In fact, when she looked across different stages of AD, she found less VAMP2 in the CSF of people who were amyloid-positive and tau-negative than in CSF of controls, but more VAMP2 in CSF of amyloid-positive/tau-positive people, i.e., those further along in their pathogenesis. The data suggest that as AD develops, CSF VAMP2 first drops, then rises. This rise correlated tightly with levels of phospho-tau in the CSF, Belbin said. Belbin found a similar nonlinear progression in Down’s syndrome (Lleó et al., 2021). 

Zetterberg called the data fascinating. “There are interplays here between α-synuclein and AD pathology, and you have to ask what role amyloid plays,” he said. Synapses contain large amounts of α-synuclein. "If synapses are exposed to amyloid, then maybe both together cause harm in a way that affects this synaptic marker,” he suggested. In fact, fluorescence resonance spectroscopy suggests that amyloid might disrupt interactions between VAMP2 and SNAP-25 (Sharda et al., 2020).

Belbin also showed that, in CSF samples from the University of Pennsylvania FTD Center and the UPenn Alzheimer Disease Research Center, VAMP2 was elevated in AD but unchanged in people who had either FTLD-tau or FTLD-TDP-43 (Cervantes González et al., 2022). “This again speaks to a role for amyloid,” said Zetterberg.

Still, Belbin thinks VAMP2 may help clinicians who treat FTD tease out whether the patient in front of them suffers from FTLD-TDP-43 or from FTLD-tau. In a separate, mass spectrometry-based, shotgun proteomics study, she identified more than 4,000 proteins in human CSF, of which more than 250 were synaptic (Lleó et al., 2019). Using a targeted mass-spectrometry assay to quantify a panel of nine of these synaptic proteins in CSF samples from UPenn, Belbin reported in Kuopio that reduced levels of calsyntenin-1 and neurexin-2a in CSF, taken while volunteers were alive, correlated with their postmortem levels of TDP-43 pathology, but not with tangles.

Belbin showed that a multi-marker proteomic panel made up of calsyntenin-1, the AMPA receptor subunit GluA4, and VAMP-2 distinguished FTLD-TDP-43 from FTLD-tau with an AUC of 0.83. This panel correlated with MMSE scores, whereas individually, none of its component markers correlated with cognition. Belbin thinks this panel might help trialists pick suitable participants for FTD clinical trials of drugs targeting tau or TDP-43, respectively. It could also serve as a surrogate for cognitive performance, she said.—Tom Fagan.

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References

Paper Citations

1. Cervantes González A, Belbin O. Fluid markers of synapse degeneration in synucleinopathies. J Neural Transm (Vienna). 2022 Feb;129(2):187-206. Epub 2022 Feb 11 PubMed.
2. Burré J, Sharma M, Tsetsenis T, Buchman V, Etherton MR, Südhof TC. Alpha-synuclein promotes SNARE-complex assembly in vivo and in vitro. Science. 2010 Sep 24;329(5999):1663-7. PubMed.
3. Agliardi C, Meloni M, Guerini FR, Zanzottera M, Bolognesi E, Baglio F, Clerici M. Oligomeric α-Syn and SNARE complex proteins in peripheral extracellular vesicles of neural origin are biomarkers for Parkinson's disease. Neurobiol Dis. 2021 Jan;148:105185. Epub 2020 Nov 18 PubMed.
4. Costa AS, Ferri E, Guerini FR, Rossi PD, Arosio B, Clerici M. VAMP2 Expression and Genotype Are Possible Discriminators in Different Forms of Dementia. Front Aging Neurosci. 2022;14:858162. Epub 2022 Mar 14 PubMed.
5. Alcolea D, Clarimón J, Carmona-Iragui M, Illán-Gala I, Morenas-Rodríguez E, Barroeta I, Ribosa-Nogué R, Sala I, Sánchez-Saudinós MB, Videla L, Subirana A, Benejam B, Valldeneu S, Fernández S, Estellés T, Altuna M, Santos-Santos M, García-Losada L, Bejanin A, Pegueroles J, Montal V, Vilaplana E, Belbin O, Dols-Icardo O, Sirisi S, Querol-Vilaseca M, Cervera-Carles L, Muñoz L, Núñez R, Torres S, Camacho MV, Carrió I, Giménez S, Delaby C, Rojas-Garcia R, Turon-Sans J, Pagonabarraga J, Jiménez A, Blesa R, Fortea J, Lleó A. The Sant Pau Initiative on Neurodegeneration (SPIN) cohort: A data set for biomarker discovery and validation in neurodegenerative disorders. Alzheimers Dement (N Y). 2019;5:597-609. Epub 2019 Oct 14 PubMed.
6. McCormack A, Keating DJ, Chegeni N, Colella A, Wang JJ, Chataway T. Abundance of Synaptic Vesicle-Related Proteins in Alpha-Synuclein-Containing Protein Inclusions Suggests a Targeted Formation Mechanism. Neurotox Res. 2019 May;35(4):883-897. Epub 2019 Feb 22 PubMed.
7. Lleó A, Carmona-Iragui M, Videla L, Fernández S, Benejam B, Pegueroles J, Barroeta I, Altuna M, Valldeneu S, Xiao MF, Xu D, Núñez-Llaves R, Querol-Vilaseca M, Sirisi S, Bejanin A, Iulita MF, Clarimón J, Blesa R, Worley P, Alcolea D, Fortea J, Belbin O. VAMP-2 is a surrogate cerebrospinal fluid marker of Alzheimer-related cognitive impairment in adults with Down syndrome. Alzheimers Res Ther. 2021 Jun 28;13(1):119. PubMed.
8. Sharda N, Pengo T, Wang Z, Kandimalla KK. Amyloid-β Peptides Disrupt Interactions Between VAMP-2 and SNAP-25 in Neuronal Cells as Determined by FRET/FLIM. J Alzheimers Dis. 2020;77(1):423-435. PubMed.
9. Cervantes González A, Irwin DJ, Alcolea D, McMillan CT, Chen-Plotkin A, Wolk D, Sirisi S, Dols-Icardo O, Querol-Vilaseca M, Illán-Gala I, Santos-Santos MA, Fortea J, Lee EB, Trojanowski JQ, Grossman M, Lleó A, Belbin O. Multimarker synaptic protein cerebrospinal fluid panels reflect TDP-43 pathology and cognitive performance in a pathological cohort of frontotemporal lobar degeneration. Mol Neurodegener. 2022 Apr 8;17(1):29. PubMed.
10. Lleó A, Núñez-Llaves R, Alcolea D, Chiva C, Balateu-Paños D, Colom-Cadena M, Gomez-Giro G, Muñoz L, Querol-Vilaseca M, Pegueroles J, Rami L, Lladó A, Molinuevo JL, Tainta M, Clarimón J, Spires-Jones T, Blesa R, Fortea J, Martínez-Lage P, Sánchez-Valle R, Sabidó E, Bayés À, Belbin O. Changes in Synaptic Proteins Precede Neurodegeneration Markers in Preclinical Alzheimer's Disease Cerebrospinal Fluid. Mol Cell Proteomics. 2019 Mar;18(3):546-560. Epub 2019 Jan 3 PubMed.

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