Microglial Expression of the Wnt Signaling Modulator DKK2 Differs between Human Alzheimer's Disease Brains and Mouse Neurodegeneration Models

Wnt signaling is crucial for synapse and cognitive function. Indeed, deficient Wnt signaling is causally related to increased expression of DKK1, an endogenous negative Wnt regulator, and synapse loss, both of which likely contribute to cognitive decline in Alzheimer's disease (AD). Increasingly, AD research efforts have probed the neuroinflammatory role of microglia, the resident immune cells of the CNS, which have furthermore been shown to be modulated by Wnt signaling. The DKK1 homolog DKK2 has been previously identified as an activated response and/or disease-associated microglia (DAM/ARM) gene in a mouse model of AD. Here, we performed a detailed analysis of DKK2 in mouse models of neurodegeneration, and in human AD brain. In APP/PS1 and APPNL-G-F AD mouse model brains as well as in SOD1G93A ALS mouse model spinal cords, but not in control littermates, we demonstrated significant microgliosis and microglial Dkk2 mRNA upregulation in a disease-stage-dependent manner. In the AD models, these DAM/ARM Dkk2+ microglia preferentially accumulated close to βAmyloid plaques. Furthermore, recombinant DKK2 treatment of rat hippocampal primary neurons blocked WNT7a-induced dendritic spine and synapse formation, indicative of an anti-synaptic effect similar to that of DKK1. In stark contrast, no such microglial DKK2 upregulation was detected in the postmortem human frontal cortex from individuals diagnosed with AD or pathologic aging. In summary, the difference in microglial expression of the DAM/ARM gene DKK2 between mouse models and human AD brain highlights the increasingly recognized limitations of using mouse models to recapitulate facets of human neurodegenerative disease.Significance StatementThe endogenous negative Wnt regulator Dkk2 is significantly upregulated at the mRNA level in microglia of Alzheimer's disease (AD) mouse models, implying that microglia derived Dkk2 protein may detrimentally contribute to a reduced Wnt signaling tone in the AD brain, a known pathophysiological manifestation. Indeed, recombinant DKK2 prevented Wnt-dependent synapse formation in cultured neurons. However, DKK2 upregulation was not recapitulated in postmortem human AD brains. The success of neurodegeneration animal models has relied on pathophysiology that for the most part correctly modelled human disease. Increasingly, however, limitations to the validity of mouse models to recapitulate human neurodegenerative disease have become apparent, as evidenced by the present study by the difference in microglial DKK2 expression between AD mouse models and human AD brain

Mutations in the intellectual disability gene Ube2a cause neuronal dysfunction and impair parkin-dependent mitophagy

The prevalence of intellectual disability is around 3%; however, the etiology of the disease remains unclear in most cases. We identified a series of patients with X-linked intellectual disability presenting mutations in the Rad6a (Ube2a) gene, which encodes for an E2 ubiquitin-conjugating enzyme. Drosophila deficient for dRad6 display defective synaptic function as a consequence of mitochondrial failure. Similarly, mouse mRad6a (Ube2a) knockout and patient-derived hRad6a (Ube2a) mutant cells show defective mitochondria. Using in vitro and in vivo ubiquitination assays, we show that RAD6A acts as an E2 ubiquitin-conjugating enzyme that, in combination with an E3 ubiquitin ligase such as Parkin, ubiquitinates mitochondrial proteins to facilitate the clearance of dysfunctional mitochondria in cells. Hence, we identify RAD6A as a regulator of Parkin-dependent mitophagy and establish a critical role for RAD6A in maintaining neuronal function

Subtle behavioral changes and increased prefrontal-hippocampal network synchronicity in APPNL-G-F mice before prominent plaque deposition

Amyloid-β (Aβ) peptides occur in the brains of patients with Alzheimer's disease (AD), but their role in functional impairment is still debated. High levels of APP and APP fragments in mice that overexpress APP might confound their use in preclinical research. We examined the occurrence of behavioral, cognitive and neuroimaging changes in APPNL-G-F knock-in mice that display Aβ42 amyloidosis in the absence of APP overexpression. Female APPNL-G-F mice (carrying Swedish, Iberian and Arctic APP mutations) were compared to APPNL mice (APP Swedish) at 3, 7 and 10 months. Mice were subjected to a test battery that referred to clinical AD symptoms, comprising cage activity, open field, elevated plus maze, social preference and novelty test, and spatial learning, reversal learning and spatial reference memory performance. Our assessment confirmed that behavior at these early ages was largely unaffected in these mice in accordance with previous reports, with some subtle behavioral changes, mainly in social and anxiety-related test performance. Resting-state functional MRI (rsfMRI) assessed connectivity between hippocampal and prefrontal regions with an established role in flexibility, learning and memory. Increased prefrontal-hippocampal network synchronicity was found in 3-month-old APPNL-G-F mice. These functional changes occurred before prominent amyloid plaque deposition

MEG3 activates necroptosis in human neuron xenografts modeling Alzheimer’s disease

Neuronal cell loss is a defining feature of Alzheimer’s disease (AD), but the underlying mechanisms remain unclear. We xenografted human or mouse neurons into the brain of a mouse model of AD. Only human neurons displayed tangles, Gallyas silver staining, granulovacuolar neurodegeneration (GVD), phosphorylated tau blood biomarkers, and considerable neuronal cell loss. The long noncoding RNA MEG3 was strongly up-regulated in human neurons. This neuron-specific long noncoding RNA is also up-regulated in AD patients. MEG3 expression alone was sufficient to induce necroptosis in human neurons in vitro. Down-regulation of MEG3 and inhibition of necroptosis using pharmacological or genetic manipulation of receptor-interacting protein kinase 1 (RIPK1), RIPK3, or mixed lineage kinase domain-like protein (MLKL) rescued neuronal cell loss in xenografted human neurons. This model suggests potential therapeutic approaches for AD and reveals a human-specific vulnerability to AD

Astrocyte calcium dysfunction causes early network hyperactivity in Alzheimer's disease

Dysfunctions of network activity and functional connectivity (FC) represent early events in Alzheimer's disease (AD), but the underlying mechanisms remain unclear. Astrocytes regulate local neuronal activity in the healthy brain, but their involvement in early network hyperactivity in AD is unknown. We show increased FC in the human cingulate cortex several years before amyloid deposition. We find the same early cingulate FC disruption and neuronal hyperactivity in AppNL-F mice. Crucially, these network disruptions are accompanied by decreased astrocyte calcium signaling. Recovery of astrocytic calcium activity normalizes neuronal hyperactivity and FC, as well as seizure susceptibility and day/night behavioral disruptions. In conclusion, we show that astrocytes mediate initial features of AD and drive clinically relevant phenotypes

Early alterations in the MCH system link aberrant neuronal activity and sleep disturbances in a mouse model of Alzheimer’s disease

Early Alzheimer’s disease (AD) is associated with hippocampal hyperactivity and decreased sleep quality. Here we show that homeostatic mechanisms transiently counteract the increased excitatory drive to CA1 neurons in App NL-G-F mice, but that this mechanism fails in older mice. Spatial transcriptomics analysis identifies Pmch as part of the adaptive response in App NL-G-F mice. Pmch encodes melanin-concentrating hormone (MCH), which is produced in sleep–active lateral hypothalamic neurons that project to CA1 and modulate memory. We show that MCH downregulates synaptic transmission, modulates firing rate homeostasis in hippocampal neurons and reverses the increased excitatory drive to CA1 neurons in App NL-G-F mice. App NL-G-F mice spend less time in rapid eye movement (REM) sleep. App NL-G-F mice and individuals with AD show progressive changes in morphology of CA1-projecting MCH axons. Our findings identify the MCH system as vulnerable in early AD and suggest that impaired MCH-system function contributes to aberrant excitatory drive and sleep defects, which can compromise hippocampus-dependent functions

Gamma-secretase activity requires the presenilin dependent trafficking of nicastrin through the Golgi apparatus but not its complex glycosylation

Nicastrin and presenilin are two major components of the gamma-secretase complex, which executes the intramembrane proteolysis of type I integral membrane proteins such as the amyloid precursor protein (APP) and Notch. Nicastrin is synthesized in fibroblasts and neurons as an endoglycosidase-H-sensitive glycosylated precursor protein (immature nicastrin) and is then modified by complex glycosylation in the Golgi apparatus and by sialylation in the trans-Golgi network (mature nicastrin). These modifications are not observed with exogenously overexpressed nicastrin. Under normal cell culture conditions, only mature nicastrin is expressed at the cell surface and binds to the presenilin heterodimers. Mature nicastrin has a half-life of more than 24 hours. In the absence of presenilin 1 and 2, nicastrin remains entirely endoglycosidase H sensitive, is retained in the endoplasmic reticulum and is slowly degraded. Single presenilin 1 or presenilin 2 deficiency affects glycosylation of nicastrin to a lesser extent than the combined presenilin deficiencies, suggesting a correlation between either the transport of nicastrin out of the endoplasmic reticulum or the concomitant complex glycosylation of nicastrin, and gamma-secretase activity. However, when complex glycosylation of nicastrin was inhibited using mannosidase I inhibitors, gamma-secretase cleavage of APP or Notch was not inhibited and the immature nicastrin still associates with presenilin and appears at the cell surface. Complex glycosylation of nicastrin is therefore not needed for gamma-secretase activity. Because the trafficking of nicastrin to the Golgi apparatus is dependent on presenilins, our data point to a central role of presenilin in nicastrin maturation/localization, which could help to partially resolve the 'spatial paradox'