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Alzheimer’s disease (AD) has been associated with cardiovascular and cerebrovascular risk factors (CVRFs) during middle age and later and is frequently accompanied by cerebrovascular pathology at death. An interaction between CVRFs and genetic variants might explain the pathogenesis. Genome-wide, gene by CVRF interaction analyses for AD, in 6568 patients and 8101 controls identified FMNL2 (p = 6.6 × 10–7). A significant increase in FMNL2 expression was observed in the brains of patients with brain infarcts and AD pathology and was associated with amyloid and phosphorylated tau deposition. FMNL2 was also prominent in astroglia in AD among those with cerebrovascular pathology. Amyloid toxicity in zebrafish increased fmnl2a expression in astroglia with detachment of astroglial end feet from blood vessels. Knockdown of fmnl2a prevented gliovascular remodeling, reduced microglial activity and enhanced amyloidosis. APP/PS1dE9 AD mice also displayed increased Fmnl2 expression and reduced the gliovascular contacts independent of the gliotic response. Based on this work, we propose that FMNL2 regulates pathology-dependent plasticity of the blood–brain-barrier by controlling gliovascular interactions and stimulating the clearance of extracellular aggregates. Therefore, in AD cerebrovascular risk factors promote cerebrovascular pathology which in turn, interacts with FMNL2 altering the normal astroglial-vascular mechanisms underlying the clearance of amyloid and tau increasing their deposition in brain.

Epitranscriptomic regulation adds a layer of post-transcriptional control to brain function during development and adulthood. The identification of RNA-modifying enzymes has opened the possibility of investigating the role epitranscriptomic changes play in the disease process. NOP2/Sun RNA methyltransferase 2 (NSun2) is one of the few known brain-enriched methyltransferases able to methylate mammalian non-coding RNAs. NSun2 loss of function due to autosomal-recessive mutations has been associated with neurological abnormalities in humans. Here, we show NSun2 is expressed in adult human neurons in the hippocampal formation and prefrontal cortex. Strikingly, we unravel decreased NSun2 protein expression and an increased ratio of pTau/NSun2 in the brains of patients with Alzheimer’s disease (AD) as demonstrated by Western blotting and immunostaining, respectively. In a well-established Drosophila melanogaster model of tau-induced toxicity, reduction of NSun2 exacerbated tau toxicity, while overexpression of NSun2 partially abrogated the toxic effects. Conditional ablation of NSun2 in the mouse brain promoted a decrease in the miR-125b m6A levels and tau hyperphosphorylation. Utilizing human induced pluripotent stem cell (iPSC)-derived neuronal cultures, we confirmed NSun2 deficiency results in tau hyperphosphorylation. We also found that neuronal NSun2 levels decrease in response to amyloid-beta oligomers (AβO). Notably, AβO-induced tau phosphorylation and cell toxicity in human neurons could be rescued by overexpression of NSun2. Altogether, these results indicate that neuronal NSun2 deficiency promotes dysregulation of miR-125b and tau phosphorylation in AD and highlights a novel avenue for therapeutic targeting.

In this chapter, we present the methodology currently used in our laboratory to generate a starPEG-MMP (starPEG)- and heparin maleimide HM06 (heparin)-based 3D cell culture system, in a hydrogel, that can be used to study human neuronal development and Alzheimer's disease (AD) pathology. A 3D cell culture system can mimic the in vivo cellular environment better than a 2D format, in which these cells exhibit neural network formation, electrophysiological activity, tissue-specific extracellular matrix (ECM) deposition, and neurotransmitter responsiveness. When treated with amyloid beta-42 (Aβ42) peptides, this system recapitulates many of the pathological effects of AD, including reduced neural stem cell proliferation, impaired neuronal network formation, dystrophic axonal ends, synaptic loss, failure to deposit ECM, elevated tau hyperphosphorylation, and formation of neurofibrillary tangles. Culturing human primary cortical astrocyte (pHA)- or induced pluripotent stem cell (iPSC)-derived human neural stem cells in this biohybrid hydrogel system has led to the discovery of novel regulatory pathways underlying neurodegenerative pathology in different phases of AD.

Neurogenesis relates to the brain resilience and is reduced in Alzheimer’s disease (AD). Restoring healthy levels of neurogenesis could have beneficial effects for coping with neurodegeneration. However, molecular mechanisms that could enhance neurogenesis from astroglial progenitors in AD pathology are largely unknown. We used lentiviruses to expressNgfrin the hippocampus of the APP/PS1dE9 mouse model of AD, histologically analyzed the changes in proliferation of neural stem cells and neurogenesis; performed single-cell transcriptomics, spatial proteomics, and functional knockdown studies. We found that expression ofNgfr, a neurogenic determinant in pathology-induced neuroregeneration in zebrafish, stimulated proliferative and neurogenic outcome in the APP/PS1dE9 AD mouse model. Ngfr suppressed reactive astrocyte marker Lipocalin-2 (Lcn2) in astroglia. Blockage of Lcn2 receptor, Slc22a17, recapitulated the neurogenic effects of NGFR, and long-term Ngfr expression reduced amyloid plaques and Tau phosphorylation. Furthermore, immunostaining on postmortem human hippocampi with AD or primary age-related Tauopathy and 3D human astroglial cultures showed that elevated LCN2 levels correlate with gliosis. By comparing transcriptional changes in mouse hippocampus, zebrafish brain, and human AD brains in terms of cell intrinsic differential gene expression analyses as well as weighted gene co-expression network analysis, we observed common potential downstream effectors of NGFR signaling,C4BandPFKP, that are relevant to AD. Our study links the regulatory role of an autocrine molecular mechanism in astroglia to the neurogenic ability and modulatory effects on amyloid and tau phosphorylation, opening new research avenues and suggesting that neurogenesis-oriented therapeutic approaches could be a potential clinical intervention for AD.

Neurogenesis decreases in Alzheimer’s disease (AD) patients, suggesting that restoring the normal neurogenic response could be a disease modifying intervention. To study the mechanisms of pathology-induced neuro-regeneration in vertebrate brains, zebrafish is an excellent model due to its extensive neural regeneration capacity. Here, we report that Kynurenic acid (KYNA), a metabolite of the amino acid tryptophan, negatively regulates neural stem cell (NSC) plasticity in adult zebrafish brain through its receptor, aryl hydrocarbon receptor 2 (Ahr2). The production of KYNA is suppressed after amyloid-toxicity through reduction of the levels of Kynurenine amino transferase 2 (KAT2), the key enzyme producing KYNA. NSC proliferation is enhanced by an antagonist for Ahr2 and is reduced with Ahr2 agonists or KYNA. A subset of Ahr2-expressing zebrafish NSCs do not express other regulatory receptors such as il4r or ngfra, indicating that ahr2-positive NSCs constitute a new subset of neural progenitors that are responsive to amyloid-toxicity. By performing transcriptome-wide association studies (TWAS) in three late onset Alzheimer disease (LOAD) brain autopsy cohorts, we also found that several genes that are components of KYNA metabolism or AHR signaling are differentially expressed in LOAD, suggesting a strong link between KYNA/Ahr2 signaling axis to neurogenesis in LOAD.

Recent findings suggest that reduced neurogenesis could be one of the underlying reasons for the exacerbated neuropathology in humans, thus restoring the neural stem cell proliferation and neurogenesis could help to circumvent some pathological aspects of Alzheimer's disease. We recently identified Interleukin-4/STAT6 signaling as a neuron-glia crosstalk mechanism that enables glial proliferation and neurogenesis in adult zebrafish brain and 3D cultures of human astroglia, which manifest neurogenic properties. In this study, by using single cell sequencing in the APP/PS1dE9 mouse model of AD, we found that IL4 receptor (Il4r) is not expressed in mouse astroglia and IL4 signaling is not active in these cells. We tested whether activating IL4/STAT6 signaling would enhance cell proliferation and neurogenesis in healthy and disease conditions. Lentivirus-mediated expression of IL4R or constitutively active STAT6VT impaired the survival capacity of mouse astroglia in vivo but not in vitro. These results suggest that the adult mouse brain generates a non-permissive environment that dictates a negative effect of IL4 signaling on astroglial survival and neurogenic properties in contrast to zebrafish brains and in vitro mammalian cell cultures. Our findings that IL4R signaling in dentate gyrus (DG) of adult mouse brain impinges on the survival of DG cells implicate an evolutionary mechanism that might underlie the loss of neuroregenerative ability of the brain, which might be utilized for basic and clinical aspects for neurodegenerative diseases.

Astrocytes are abundant cell types in the vertebrate central nervous system and can act as neural stem cells in specialized niches where they constitutively generate new neurons. Outside the stem cell niches, however, these glial cells are not neurogenic. Although injuries in the mammalian central nervous system lead to profound proliferation of astrocytes, which cluster at the lesion site to form a gliotic scar, neurogenesis does not take place. Therefore, a plausible regenerative therapeutic option is to coax the endogenous reactive astrocytes to a pre-neurogenic progenitor state and use them as an endogenous reservoir for repair. However, little is known on the mechanisms that promote the neural progenitor state after injuries in humans. Gata3 was previously found to be a mechanism that zebrafish brain uses to injury-dependent induction of neural progenitors. However, the effects of GATA3 in human astrocytes after injury are not known. Therefore, in this report, we investigated how overexpression of GATA3 in primary human astrocytes would affect the neurogenic potential before and after injury in 2D and 3D cultures. We found that primary human astrocytes are unable to induce GATA3 after injury. Lentivirus-mediated overexpression of GATA3 significantly increased the number of GFAP/SOX2 double positive astrocytes and expression of pro-neural factor ASCL1, but failed to induce neurogenesis, suggesting that GATA3 is required for enhancing the neurogenic potential of primary human astrocytes and is not sufficient to induce neurogenesis alone.