Amylin, a pancreatic β-cell-derived peptide hormone, forms inclusions in brain microvessels of patients with dementia who have been diagnosed with type 2 diabetes and Alzheimer’s disease. The ...cellular localization of these inclusions and the consequences thereof are not yet known. Using immunohistochemical staining of hippocampus and parahippocampal cortex from patients with Alzheimer’s disease and non-demented controls, we show that amylin cell inclusions are found in pericytes. The number of amylin cell inclusions did not differ between patients with Alzheimer’s disease and controls, but amylin-containing pericytes displayed nuclear changes associated with cell death and reduced expression of the pericyte marker neuron-glial antigen 2. The impact of amylin on pericyte viability was further demonstrated in in vitro studies, which showed that pericyte death increased in presence of fibril- and oligomer amylin. Furthermore, oligomer amylin increased caspase 3/7 activity, reduced lysate neuron-glial antigen 2 levels and impaired autophagy. Our findings contribute to increased understanding of how aggregated amylin affects brain vasculature and highlight amylin as a potential factor involved in microvascular pathology in dementia progression.
Previous studies have used immunohistology to demonstrate Alzheimer's disease (AD) characteristic accumulation of amyloid-β (Aβ) in the retina of AD patients, a finding indicating retina examination ...as a potential diagnostic tool for AD pathology.
To further explore this idea by investigating whether levels of Aβ42 and Aβ40 in retina are associated with corresponding levels in hippocampus, neuropathological assessments, apolipoprotein E (APOE) genotype, and levels of islet amyloid polypeptide (IAPP).
Levels of high molecular weight (HMW) Aβ42, Aβ40, and IAPP in ultra-centrifuged homogenates of retina and hippocampus from patients with AD, multiple sclerosis, AD with Lewy bodies, and non-demented controls were analyzed using Mesoscale Discovery electrochemiluminescence technology employing immunoassay and enzyme-linked immunosorbent assay.
Higher levels of retinal and hippocampal Aβ42-HMW, Aβ40-HMW, and IAPP-HMW were found in individuals with high neuropathological scores of Aβ plaques and in individuals carrying the APOEɛ4 allele. The retinal levels of Aβ42-HMW and Aβ40-HMW correlated with corresponding levels in hippocampus as well as with neurofibrillary tangles (NFT) and Aβ scores. Retinal IAPP-HMW correlated with retinal levels of Aβ42-HMW and with NFT and Aβ scores.
These results show that different isoforms of Aβ can be detected in the human retina and moreover support the growing number of studies indicating that AD-related pathological changes occurring in the brain could be reflected in the retina.
Abstract Blood-brain barrier (BBB) dysfunction might be an important component of many neurodegenerative disorders. In this study, we investigated its role in dementia using large clinical cohorts. ...The cerebrospinal fluid (CSF)/plasma albumin ratio (Qalb), an indicator of BBB (and blood-CSF barrier) permeability, was measured in a total of 1015 individuals. The ratio was increased in patients with Alzheimer’s disease (AD), dementia with Lewy bodies (DLB) or Parkinson’s disease dementia (PDD), subcortical vascular dementia (VaD) and frontotemporal dementia (FTD) compared with controls. However, this measure was not changed during preclinical or prodromal AD and was not associated with amyloid PET or APOE genotype. The Qalb was increased in diabetes mellitus and correlated positively with CSF biomarkers of angiogenesis and endothelial dysfunction (VEGF, ICAM-1 and VCAM-1). In healthy elderly, high body mass index and waist-hip ratio predicted increased Qalb 20 years later. In summary, BBB permeability is increased in major dementia disorders, but does not relate to amyloid pathology or APOE genotype. Instead, BBB impairment may be associated with diabetes and brain microvascular damage.
Reduced glucose metabolism and formation of polyglucosan bodies (PGB) are, beside amyloid beta plaques and neurofibrillary tangles, well‐known pathological findings associated with Alzheimer's ...disease (AD). Since both glucose availability and PGB are regulated by enzymatic degradation of glycogen, we hypothesize that dysfunctional glycogen degradation is a critical event in AD progression. We therefore investigated whether alpha (α)‐amylase, an enzyme known to efficiently degrade polysaccharides in the gastrointestinal tract, is expressed in the hippocampal CA1/subiculum and if the expression is altered in AD patients. Using immunohistochemical staining techniques, we show the presence of the α‐amylase isotypes AMY1A and AMY2A in neuronal dendritic spines, pericytes and astrocytes. Moreover, AD patients showed reduced gene expression of α‐amylase, but conversely increased protein levels of α‐amylase as well as increased activity of the enzyme compared with non‐demented controls. Lastly, we observed increased, albeit not significant, load of periodic acid‐Schiff positive PGB in the brain of AD patients, which correlated with increased α‐amylase activity. These findings show that α‐amylase is expressed and active in the human brain, and suggest the enzyme to be affected, alternatively play a role, in the neurodegenerative Alzheimer's disease pathology.
Islet amyloid polypeptide (IAPP) forms toxic aggregates in the brain of patients with Alzheimer's disease (AD). Whether IAPP also affects the retina in these patients is still unknown. Levels of IAPP ...in soluble and insoluble homogenate fractions of retina and hippocampus from AD patients and nondemented controls were analyzed using ELISA. Number of pericytes and vessel length were determined by analysis of immunostained retina and hippocampus. Insoluble retinal fractions of AD patients contained lower levels of unmodified IAPP, whereas soluble retinal fractions contained increased levels of the same. Total IAPP levels and pericyte numbers in retina mirrored corresponding variables in the hippocampus. Moreover, levels of total unmodified IAPP correlated negatively with the vessel length both in retina and hippocampus across the group and positively with pericyte numbers in retina in AD patients. Our studies indicate that changes in brain IAPP are reflected by corresponding levels in the retina. Our results also suggest modification of IAPP as an important event implicated in vascular changes associated with AD.
Alzheimer’s disease (AD) is a progressive neurodegenerative disease in which the formation of extracellular aggregates of amyloid beta (Aβ) peptide, fibrillary tangles of intraneuronal tau and ...microglial activation are major pathological hallmarks. One of the key molecules involved in microglial activation is galectin-3 (gal3), and we demonstrate here for the first time a key role of gal3 in AD pathology. Gal3 was highly upregulated in the brains of AD patients and 5xFAD (familial Alzheimer’s disease) mice and found specifically expressed in microglia associated with Aβ plaques. Single-nucleotide polymorphisms in the
LGALS3
gene, which encodes gal3, were associated with an increased risk of AD. Gal3 deletion in 5xFAD mice attenuated microglia-associated immune responses, particularly those associated with TLR and TREM2/DAP12 signaling. In vitro data revealed that gal3 was required to fully activate microglia in response to fibrillar Aβ. Gal3 deletion decreased the Aβ burden in 5xFAD mice and improved cognitive behavior. Interestingly, a single intrahippocampal injection of gal3 along with Aβ monomers in WT mice was sufficient to induce the formation of long-lasting (2 months) insoluble Aβ aggregates, which were absent when gal3 was lacking. High-resolution microscopy (stochastic optical reconstruction microscopy) demonstrated close colocalization of gal3 and TREM2 in microglial processes, and a direct interaction was shown by a fluorescence anisotropy assay involving the gal3 carbohydrate recognition domain. Furthermore, gal3 was shown to stimulate TREM2–DAP12 signaling in a reporter cell line. Overall, our data support the view that gal3 inhibition may be a potential pharmacological approach to counteract AD.
The present work addressed the hypothesis that NG2/CSPG4, CD146/MCAM, and VAP1/AOC3 are target genes of myocardin-related transcription factors (MRTFs: myocardin/MYOCD, MRTF-A/MKL1, MRTF-B/MKL2) and ...serum response factor (SRF). Using a bioinformatics approach, we found that CSPG4, MCAM, and AOC3 correlate with MYOCD, MRTF-A/MKL1, and SRF across human tissues. No other transcription factor correlated as strongly with these transcripts as SRF. Overexpression of MRTFs increased both mRNA and protein levels of CSPG4, MCAM, and AOC3 in cultured human smooth muscle cells (SMCs). Imaging confirmed increased staining for CSPG4, MCAM, and AOC3 in MRTF-A/MKL1-transduced cells. MRTFs exert their effects through SRF, and the MCAM and AOC3 gene loci contained binding sites for SRF. SRF silencing reduced the transcript levels of these genes, and time-courses of induction paralleled the direct target ACTA2. MRTF-A/MKL1 increased the activity of promoter reporters for MCAM and AOC3, and transcriptional activation further depended on the chromatin remodeling enzyme KDM3A. CSPG4, MCAM, and AOC3 responded to the MRTF-SRF inhibitor CCG-1423, to actin dynamics, and to ternary complex factors. Coincidental detection of these proteins should reflect MRTF-SRF activity, and beyond SMCs, we observed co-expression of CD146/MCAM, NG2/CSPG4, and VAP1/AOC3 in pericytes and endothelial cells in the human brain. This work identifies highly responsive vascular target genes of MRTF-SRF signaling that are regulated via a mechanism involving KDM3A.
The population of brain pericytes, a cell type important for vessel stability and blood brain barrier function, has recently been shown altered in patients with Alzheimer's disease (AD). The ...underlying reason for this alteration is not fully understood, but progressive accumulation of the AD characteristic peptide amyloid-beta (A beta) has been suggested as a potential culprit. In the current study, we show reduced number of hippocampal NG2+ pericytes and an association between NG2+ pericyte numbers and A1-40 levels in AD patients. We further demonstrate, using invitro studies, an aggregation-dependent impact of A beta 1-40 on human NG2+ pericytes. Fibril-EP A beta 1-40 exposure reduced pericyte viability and proliferation and increased caspase 3/7 activity. Monomer A beta 1-40 had quite the opposite effect: increased pericyte viability and proliferation and reduced caspase 3/7 activity. Oligomer-EP A beta 1-40 had no impact on either of the cellular events. Our findings add to the growing number of studies suggesting a significant impact on pericytes in the brains of AD patients and suggest different aggregation forms of A beta 1-40 as potential key regulators of the brain pericyte population size.
Neurosin is a protease that in vitro degrades α-synuclein, the main constituent of Lewy bodies found in brains of patients with synucleinopathy including Parkinson's disease (PD) and dementia with ...Lewy bodies (DLB). Several studies have reported reduced cerebrospinal fluid (CSF) levels of α-synuclein in synucleinopathy patients and recent data also proposes a significant role of α-synuclein in the pathophysiology of Alzheimer's disease (AD). To investigate potential links between neurosin and its substrate α-synuclein in vivo we used a commercially available sandwich ELISA and an in-house developed direct ELISA to quantify CSF levels of α-synuclein and neurosin in patients diagnosed with DLB, PD and PD dementia (PDD) versus AD patients and non-demented controls. We found that patients with synucleinopathy displayed lower CSF levels of neurosin and α-synuclein compared to controls and AD patients. In contrast, AD patients demonstrated significantly increased CSF α-synuclein but similar neurosin levels compared to non-demented controls. Further, CSF neurosin and α-synuclein concentrations were positively associated in controls, PD and PDD patients and both proteins were highly correlated to CSF levels of phosphorylated tau in all investigated groups. We observed no effect of gender or presence of the apolipoprotein Eε4 allele on neither neurosin or α-synuclein CSF levels. In concordance with the current literature our study demonstrates decreased CSF levels of α-synuclein in synucleinopathy patients versus AD patients and controls. Importantly, decreased α-synuclein levels in patients with synucleinopathy appear linked to low levels of the α-synuclein cleaving enzyme neurosin. In contrast, elevated levels of α-synuclein in AD patients were not related to any altered CSF neurosin levels. Thus, altered CSF levels of α-synuclein and neurosin in patients with synucleinopathy versus AD may not only mirror disease-specific neuropathological mechanisms but may also serve as fit candidates for future biomarker studies aiming at identifying specific markers of synucleinopathy.
Recent studies indicate a crucial role for neuronal glycogen storage and degradation in memory formation. We have previously identified alpha‐amylase (α‐amylase), a glycogen degradation enzyme, ...located within synaptic‐like structures in CA1 pyramidal neurons and shown that individuals with a high copy number variation of α‐amylase perform better on the episodic memory test. We reported that neuronal α‐amylase was absent in patients with Alzheimer's disease (AD) and that this loss corresponded to increased AD pathology. In the current study, we verified these findings in a larger patient cohort and determined a similar reduction in α‐amylase immunoreactivity in the molecular layer of hippocampus in AD patients. Next, we demonstrated reduced α‐amylase concentrations in oligomer amyloid beta 42 (Aβ42) stimulated SH‐SY5Y cells and neurons derived from human‐induced pluripotent stem cells (hiPSC) with PSEN1 mutation. Reduction of α‐amylase production and activity, induced by siRNA and α‐amylase inhibitor Tendamistat, respectively, was further shown to enhance glycogen load in SH‐SY5Y cells. Both oligomer Aβ42 stimulated SH‐SY5Y cells and hiPSC neurons with PSEN1 mutation showed, however, reduced load of glycogen. Finally, we demonstrate the presence of α‐amylase within synapses of isolated primary neurons and show that inhibition of α‐amylase activity with Tendamistat alters neuronal activity measured by calcium imaging. In view of these findings, we hypothesize that α‐amylase has a glycogen degrading function within synapses, potentially important in memory formation. Hence, a loss of α‐amylase, which can be induced by Aβ pathology, may in part underlie the disrupted memory formation seen in AD patients.
Alpha‐amylase and glycogen are found in neuronal synapses. Inhibition of alpha‐amylase activity with Tendamistat increases neuronal glycogen load and causes calcium dyshomeostasis. Presence of amyloid beta is associated with reduced alpha‐amylase and glycogen load.
PDF
