Cerebrospinal fluid profile of NPTX2 supports role of Alzheimer's disease-related inhibitory circuit dysfunction in adults with down syndrome

Alzheimer's disease (AD) is the major cause of death in adults with Down syndrome (DS). There is an urgent need for objective markers of AD in the DS population to improve early diagnosis and monitor disease progression. NPTX2 has recently emerged as a promising cerebrospinal fluid (CSF) biomarker of Alzheimer-related inhibitory circuit dysfunction in sporadic AD patients. The objective of this study was to evaluate NPTX2 in the CSF of adults with DS and to explore the relationship of NPTX2 to CSF levels of the PV interneuron receptor, GluA4, and existing AD biomarkers (CSF and neuroimaging). This is a cross-sectional, retrospective study of adults with DS with asymptomatic AD (aDS, n = 49), prodromal AD (pDS, n = 18) and AD dementia (dDS, n = 27). Non-trisomic controls (n = 34) and patients with sporadic AD dementia (sAD, n = 40) were included for comparison. We compared group differences in CSF NPTX2 according to clinical diagnosis and degree of intellectual disability. We determined the relationship of CSF NPTX2 levels to age, cognitive performance (CAMCOG, free and cued selective reminding, semantic verbal fluency), CSF levels of a PV-interneuron marker (GluA4) and core AD biomarkers; CSF Aβ1-42, CSF t-tau, cortical atrophy (magnetic resonance imaging) and glucose metabolism ([18F]-fluorodeoxyglucose positron emission tomography). Compared to controls, mean CSF NPTX2 levels were lower in DS at all AD stages; aDS (0.6-fold, adj.p 0.07). Low CSF NPTX2 levels were associated with low GluA4 in all clinical groups; controls (r 2 = 0.2, p = 0.003), adults with DS (r 2 = 0.4, p 0.3, p 0.3, p < 0.001), increased cortical atrophy (p < 0.05) and reduced glucose metabolism (p < 0.05). Low levels of CSF NPTX2, a protein implicated in inhibitory circuit function, is common to sporadic and genetic forms of AD. CSF NPTX2 represents a promising CSF surrogate marker of early AD-related changes in adults with DS

Changes in synaptic proteins precede neurodegeneration markers in preclinical Alzheimer's disease cerebrospinal fluid

Altres ajuts: Additional funding came from the "Programa 1 Enfermedad de Alzheimer y otras demencias degenerativas" from the Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas (CIBERNED), the "Fundació Bancaria La Caixa" (4560/6393) and "La Marató" organized by the television channel, TV3 (201426 10).A biomarker of synapse loss, an early event in Alzheimer's disease (AD) pathophysiology that precedes neuronal death and symptom onset, would be a much-needed prognostic biomarker. With direct access to the brain interstitial fluid, the cerebrospinal fluid (CSF) is a potential source of synapse-derived proteins. In this study, we aimed to identify and validate novel CSF biomarkers of synapse loss in AD. Discovery: Combining shotgun proteomics of the CSF with an exhaustive search of the literature and public databases, we identified 251 synaptic proteins, from which we selected 22 for further study. Verification: Twelve proteins were discarded because of poor detection by Selected Reaction Monitoring (SRM). We confirmed the specific expression of 9 of the remaining proteins (Calsyntenin-1, GluR2, GluR4, Neurexin-2A, Neurexin-3A, Neuroligin-2, Syntaxin-1B, Thy-1, Vamp-2) at the human synapse using Array Tomography microscopy and biochemical fractionation methods. Exploration: Using SRM, we monitored these 9 synaptic proteins (20 peptides) in a cohort of CSF from cognitively normal controls and subjects in the pre-clinical and clinical AD stages (n 80). Compared with controls, peptides from 8 proteins were elevated 1.3 to 1.6-fold (p < 0.04) in prodromal AD patients. Validation: Elevated levels of a GluR4 peptide at the prodromal stage were replicated (1.3-fold, p 0.04) in an independent cohort (n 60). Moreover, 7 proteins were reduced at preclinical stage 1 (0.6 to 0.8-fold, p < 0.04), a finding that was replicated (0.7 to 0.8-fold, p < 0.05) for 6 proteins in a third cohort (n 38). In a cross-cohort meta-analysis, 6 synaptic proteins (Calsyn-tenin-1, GluR4, Neurexin-2A, Neurexin-3A, Syntaxin-1B and Thy-1) were reduced 0.8-fold (p < 0.05) in preclinical AD, changes that precede clinical symptoms and CSF markers of neurodegeneration. Therefore, these proteins could have clinical value for assessing disease progression, especially in preclinical stages of AD

VAMP-2 is a surrogate cerebrospinal fluid marker of Alzheimer-related cognitive impairment in adults with Down syndrome

Altres ajuts: Fundació la Marató de TV3/20141210There is an urgent need for objective markers of Alzheimer's disease (AD)-related cognitive impairment in people with Down syndrome (DS) to improve diagnosis, monitor disease progression, and assess response to disease-modifying therapies. Previously, GluA4 and neuronal pentraxin 2 (NPTX2) showed limited potential as cerebrospinal fluid (CSF) markers of cognitive impairment in adults with DS. Here, we compare the CSF profile of a panel of synaptic proteins (Calsyntenin-1, Neuroligin-2, Neurexin-2A, Neurexin-3A, Syntaxin-1B, Thy-1, VAMP-2) to that of NPTX2 and GluA4 in a large cohort of subjects with DS across the preclinical and clinical AD continuum and explore their correlation with cognitive impairment. We quantified the synaptic panel proteins by selected reaction monitoring in CSF from 20 non-trisomic cognitively normal controls (mean age 44) and 80 adults with DS grouped according to clinical AD diagnosis (asymptomatic, prodromal AD or AD dementia). We used regression analyses to determine CSF changes across the AD continuum and explored correlations with age, global cognitive performance (CAMCOG), episodic memory (modified cued-recall test; mCRT) and CSF biomarkers, CSF Aβ ratio, CSF Aβ, CSF p-tau, and CSF NFL. P values were adjusted for multiple testing. In adults with DS, VAMP-2 was the only synaptic protein to correlate with episodic memory (delayed recall adj.p =.04) and age (adj.p =.0008) and was the best correlate of CSF Aβ (adj.p =.0001), p-tau (adj.p < .0001), and NFL (adj.p < .0001). Compared to controls, mean VAMP-2 levels were lower in asymptomatic adults with DS only (adj.p =.02). CSF levels of Neurexin-3A, Thy-1, Neurexin-2A, Calysntenin-1, Neuroligin-2, GluA4, and Syntaxin-1B all strongly correlated with NPTX2 (p <.0001), which was the only synaptic protein to show reduced CSF levels in DS at all AD stages compared to controls (adj.p <.002). These data show proof-of-concept for CSF VAMP-2 as a potential marker of synapse degeneration that correlates with CSF AD and axonal degeneration markers and cognitive performance

Plasma extracellular vesicles reveal early molecular differences in amyloid positive patients with early-onset mild cognitive impairment

In the clinical course of Alzheimer's disease (AD) development, the dementia phase is commonly preceded by a prodromal AD phase, which is mainly characterized by reaching the highest levels of Aβ and p-tau-mediated neuronal injury and a mild cognitive impairment (MCI) clinical status. Because of that, most AD cases are diagnosed when neuronal damage is already established and irreversible. Therefore, a differential diagnosis of MCI causes in these prodromal stages is one of the greatest challenges for clinicians. Blood biomarkers are emerging as desirable tools for pre-screening purposes, but the current results are still being analyzed and much more data is needed to be implemented in clinical practice. Because of that, plasma extracellular vesicles (pEVs) are gaining popularity as a new source of biomarkers for the early stages of AD development. To identify an exosome proteomics signature linked to prodromal AD, we performed a cross-sectional study in a cohort of early-onset MCI (EOMCI) patients in which 184 biomarkers were measured in pEVs, cerebrospinal fluid (CSF), and plasma samples using multiplex PEA technology of Olink© proteomics. The obtained results showed that proteins measured in pEVs from EOMCI patients with established amyloidosis correlated with CSF p-tau181 levels, brain ventricle volume changes, brain hyperintensities, and MMSE scores. In addition, the correlations of pEVs proteins with different parameters distinguished between EOMCI Aβ( +) and Aβ(-) patients, whereas the CSF or plasma proteome did not. In conclusion, our findings suggest that pEVs may be able to provide information regarding the initial amyloidotic changes of AD. Circulating exosomes may acquire a pathological protein signature of AD before raw plasma, becoming potential biomarkers for identifying subjects at the earliest stages of AD development

Clonal Astrocytic Response to Cortical Injury

<div><p>Astrocytes are a heterogeneous population of glial cells with multifaceted roles in the central nervous system. Recently, the new method for the clonal analysis Star Track evidenced the link between astrocyte heterogeneity and lineage. Here, we tested the morphological response to mechanical injury of clonally related astrocytes using the Star Track approach, which labels each cell lineage with a specific code of colors. Histological and immunohistochemical analyses at 7 days post injury revealed a variety of morphological changes that were different among distinct clones. In many cases, cells of the same clone responded equally to the injury, suggesting the dependence on their genetic codification (intrinsic response). However, in other cases cells of the same clone responded differently to the injury, indicating their response to extrinsic factors. Thus, whereas some clones exhibited a strong morphological alteration or a high proliferative response to the injury, other clones located at similar distances to the lesion were apparently unresponsive. Concurrence of different clonal responses to the injury reveals the importance of the development determining the astrocyte features in response to brain injuries. These features should be considered to develop therapies that affect glial function.</p></div

Cell proliferation detected by iMmunohistochemistry against pH 3 and Ki67 proliferative markers at seven days post injury.

<p><b>A.</b> Low magnification of the injured hemisphere showing the merge of the Star Track staining with the labeling against the proliferative marker pH<b>B.</b> pH 3 labeling of the image in (A). <b>C–F.</b> High magnifications of the regions highlighted in A–B. <b>C–E</b> PH 3 labeling, <b>D–F</b> pH 3 channel merged with the Star Track labeling. <b>G, I, K, M.</b> Labeling of the Ki67 inmunohistochemistry. <b>H, J, L, N.</b> Merging of the Star Track and Ki67 labeling. Blue circles indicate those cells labeled by the Star Track co-expressing the proliferation markers pH 3 and Ki67. Blue circles indicate those Star Track labeled cells co-expressing proliferation markers. Scale: A–B 400 µm; C–N 100 µm.</p

Cumulative proliferation assay based on the BrdU incorporation during seven days after injury.

<p><b>A.</b>Low magnification image showing the Star Track clonal labeling. <b>B.</b> Same image showing immunohistochemistry against GFP (grey) and BrdU (red). C–F. High magnification of areas highlighted in insets of A–B. <b>G–L.</b> Additional sections labeled with Star Track and GFP/BrdU immunohistochemistry. <b>G–H.</b> Images at low magnification showing the Star Track labeling. <b>I–L.</b> High magnification images of regions highlighted in insets. For each image, cells that are part of the same clone are encircled. Thick circles represent those cells that incorporated BrdU. Scale: A–B, G–H 400 µm; C–F, I–L 50 µm.</p

Reactive astrocyte clones in the lesion area and within the needle track.

<p><b>A.</b> Nissl staining showing the damage after the cortical injury. Note the increment in cell density and cellular debris delimiting the needle track. <b>B.</b> Representative section of the astrocyte clones disposition around the injury. A bunch of astrocyte morphologies is observed around the injury with clones of reactive astrocytes close to the injury. <b>C.</b> Magnification of the box highlighted in B. <b>D.</b> Groups of hypertrofied astrocytes around the wounded area. <b>E–F.</b> Hypertrophied clones emitting their cell processes to the injury edge, full of cellular debri. <b>G.</b> Representative image of a typical protoplasmic clone located in intact cortical brain areas. These astrocytes are characterized by their round shapes. Small insets show merged and individual channels of small clonal groups containing cells that share the same fluorescent marks after electroporation with the Star Track mixture. <b>H.</b> Representative image of the fibrous hypertrophied astrocytes typical in reactive astrocytes after injury. Note the change from round shapes (G) by one more enlarged with thicker cellular processes (H). Scale bars: A, 1 mm; a, 100 µm; B, D, E 500 µm; C, F, G, H 100 µm.</p

Cortical clonal dispersion in control adult mice after electroporations at E14.

<p><b>A.</b> Representative scheme of the most anterior cortical lesion. <b>B.</b> Low magnification view of the clonal groups located at the level of the rostral coordinate chosen for the injury. The majority of clonal groups exhibit protoplasmic morphologies throughout the cortical layers. Note some clonal cells attached to the lateral ventricle with their radial processes extending from the ventricle across the corpus callosum. <b>C.</b> Detail of inset in B. <b>D.</b> Representative scheme of the posterior stereotaxic coordinate chosen for the cortical lesion. <b>E.</b> Clonal dispersion of cells with some clones attached to the pial surface with a fibroblast-morphology <b>F.</b> Detail of the inset in E. Scale bars: B,E 500 µM; C,F 200 µm.</p