Microglia facilitate loss of perineuronal nets in the Alzheimer's disease brain

May combine two claims.

Microglial activation with LPS in wild-type mice elicits perineuronal net loss, impairments in the structural integrity of PV+ interneuron-associated perineuronal nets, and reduced PV+ cell densities.

“similar phenotypes are elicited in wild-type mice following microglial activation with LPS.”

• Also stated in: § Introduction · plain

  “Inducing microglial activation via lipopolysaccharide (LPS) injection in wild-type (WT) mice is sufficient to cause overall PNN loss and impairments in the structural integrity of PV+ interneuron-associated PNNs, in addition to reduced PV+ cell densities.”

• Also stated in: § Materials and methods · plain

  “(I-J) LPS induced significant decreases in WFA+ coverage in the subiculum (p<0.05) and visual cortex of adult WT mice (p<0.01; two-tailed unpaired t-test; n = 3–4/group)”

May combine two claims.

Perineuronal nets are extensively lost in the Alzheimer's disease brain, in both mouse models and human tissue.

“we report that PNNs are extensively lost in AD in proportion to plaque burden.”

• Also stated in: § Introduction · plain

  “we report that PNNs are extensively lost in the mouse and human AD brain.”

• Also stated in: § Funding · plain

  “We find that perineuronal nets are extensively lost in disease”

May combine two claims.

Extensive age- and plaque-related perineuronal net loss occurs in the 5xFAD brain, beginning in high pathology regions at 4 months and spreading globally by 18 months.

“(A) Representative whole-brain images of WFA+ PNNs and Thio-S+ dense-core plaques reveal extensive age- and plaque-related PNN loss in the 5xFAD brain, beginning in high pathology regions (e.g. subiculum) at 4 months (mo) and spreading globally by 18mo.”

May combine two claims.

PNN loss negatively correlates with plaque number, best modeled by a linear equation in the subiculum and a nonlinear quadratic equation in the visual cortex.

“This negative correlation was modeled best by a linear equation in the subiculum (Y=−188.8*X + 23,957; r2=0.2346; Pearson's r=−0.4844, p<0.01; Fig. S1A), and a nonlinear quadratic equation in the visual cortex (Y = 57,400+−2383*X + 41.54*X2; r2=0.4131; Spearman's r=−0.6090, p<0.0001; Fig. S1B) suggesting potential region-related differences.”

May combine two claims.

Thio-S-positive plaques are substantial in the subiculum but scarce in the visual cortex at 4 months, and increase between 4 and 8 months in both regions.

“(F) Thio-S+ plaques were substantial in the subiculum but scarce in the visual cortex at 4mo, and increased between 4 and 8mo in both subiculum and cortex (p<0.001, p<0.01; 1-way ANOVAs with Tukey's post-hoc test; n = 10/group), remaining at similar levels thereafter.”

May combine two claims.

LPS treatment in WT mice induced microglial activation and significantly reduced WFA+ area in the subiculum and cortex, with reductions in mean WFA+ intensity of PV+ neurons and PV+ cell densities resembling those seen in 5xFAD mice.

“LPS induced phenotypic activation of subiculum and cortical microglia (e.g. process retraction and enlargement), and while IBA1+ numbers were significantly elevated only in the latter (p<0.05; Fig. 2G-H), WFA+ area was significantly reduced in both regions (p<0.05, p<0.01; Fig. 2I-J) with reductions in mean WFA+ intensity of PV+ neurons (p = 0.051, p<0.05; Fig. 2K-L) and PV+ cell densities (p<0.05”

May combine two claims.

Microglia are increased in number in the visual cortex and display morphological hallmarks of activation in both subiculum and cortex after LPS treatment.

“(G-H) Microglia were increased in number in the visual cortex (p<0.05; two-tailed unpaired t-test; n = 4/group) and displayed morphological hallmarks of activation in both subiculum and cortex.”

May combine two claims.

5xFAD mice had significantly higher microglial densities compared to WT at every timepoint in the subiculum, and from 8 months onward in the visual cortex.

“Indeed, 5xFAD mice had significantly higher microglial densities compared to WT at every timepoint in the subiculum, and from 8mo onward in the visual cortex (p<0.0001 in each case; Fig. 1D-E).”

May combine two claims.

WFA+ PNNs were robustly decreased in 5xFAD mice, with the largest effects in areas with the highest plaque number.

“Notably, we found that WFA+ PNNs were robustly decreased in 5xFAD mice, with the largest effects seen in the areas with the highest plaque number (Fig. 1A-C).”

May combine two claims.

Total subiculum WFA+ area was reduced at every timepoint compared to age-matched WT mice, whereas the visual cortex displayed significant losses at 8 months and every timepoint thereafter.

“Total subiculum WFA+ area was reduced at every timepoint compared to age-matched WT mice (p<0.0001 in each case; Fig. 1B,G), whereas the visual cortex displayed significant losses at 8mo and every timepoint thereafter (p<0.0001 or p<0.001 in every case; Fig. 1C,H).”

May combine two claims.

PV+ interneurons are decreased following perineuronal net loss in 5xFAD mice, and these effects are mirrored by LPS-induced microglial activation.

“PV+ interneurons are decreased following PNN loss in 5xFAD mice and these effects are mirrored by LPS-induced microglial activation.”

May combine two claims.

In 3xTg-AD mice, 1-month PLX5622 treatment significantly increased WFA+ PNN area in the subiculum and visual cortex, significantly reduced microglia, but had no effect on plaque number.

“Quantification of WFA+ area reveals significant increases in subiculum and visual cortex PNNs (p = 0.0567, p<0.05) following 1-month PLX5622, which (M-N) significantly reduced microglia in the respective regions (p<0.05, p<0.01) but (O) had no effect on plaque number (subiculum p = 0.917; two-tailed unpaired t-tests; n = 4–5/group).”

• AssumptionRests on an assumption: The 5xFAD and 3xTg-AD mouse models recapitulate key aspects of human AD pathology relevant to PNN loss and microglial mediation.
• AssumptionRests on an assumption: Microglial depletion with PLX5622 affects PNNs only through microglial loss and not through direct effects on PNNs or other cells.

May combine two claims.

No disease-related differences in PV+ cell density were detected in the anterior visual cortex at 4 or 18 months, although some loss in PV+ neuronal WFA+ intensity was evident.

“We were unable to detect any disease-related differences in PV+ cell density in the anterior visual cortex at 4mo or 18mo, although some loss in PV+ neuronal WFA+ intensity was evident (Fig. S3A-C).”

May combine two claims.

CSF1R inhibitor PLX5622 completely prevented loss of WFA+ PNNs in 5xFAD subiculum and visual cortex at 4 months, despite persistence of Thio-S+ plaques in the subiculum.

“Representative 20X images of subiculum and visual cortex in 4mo 5xFAD and WT mice treated prior to and during plaque development (1.5–4mo) with sustained delivery of CSF1R inhibitor PLX5622 (1200 ppm in chow) demonstrate complete prevention of WFA+ PNN loss with microglial depletion in 5xFAD subiculum and cortex, even despite the persistence of Thio-S+ plaques in the former.”

• AssumptionRests on an assumption: Microglial depletion with PLX5622 affects PNNs only through microglial loss and not through direct effects on PNNs or other cells.

May combine two claims.

PAM were evident around dense-core plaques, and aggrecan was present within both the plaque core and surrounding microglia, with microglial processes in close spatial association with nearby intact PNNs.

“PAM were readily evident around dense-core plaques (Fig. 3D, arrows), and closer examination revealed the ubiquitous presence of aggrecan within both the plaque core and surrounding microglia (Fig. 3H), the processes of which could be seen in close spatial association with nearby intact PNNs (Fig. 3I).”

May combine two claims.

Plaques were abundant in the 5xFAD subiculum at 4 months and significantly increased by 8 months, whereas visual cortex plaques were sparse until 8 months, then evident across cortical layers, with numbers in both regions remaining stable thereafter.

“Plaques were abundant in the 5xFAD subiculum at 4mo and significantly increased by 8mo (p = 0.0002), whereas visual cortex plaques were sparse until 8mo (p<0.01 vs 4mo) at which point they were evident across cortical layers, with numbers in both regions remaining stable thereafter (Fig. 1F).”

May combine two claims.

PV+ cell numbers remained unchanged at 4 and 8 months in the 5xFAD subiculum, but were significantly reduced at 12 and 18 months compared to WT and to 5xFAD at 4 months.

“Interestingly, PV+ cell numbers remained unchanged at 4mo and 8mo in the 5xFAD subiculum (p = 0.085 at 8mo; Fig. 2B), when PNN loss is already extensive, but were significantly reduced at 12mo and 18mo compared to WT (p<0.0001, p<0.01) and to 5xFAD at 4mo (p<0.05, p<0.05), indicating that interneurons are included among the general neuronal loss that we and others have reported in the diseased sub”

May combine two claims.

Age-dependent PNN reductions occurred in both genotypes and regions, with greater PNN loss in 5xFAD; WFA+ PNNs were significantly reduced by 12 months and 8 months in diseased subiculum and visual cortex, respectively, compared to the same 5xFAD regions at 4 months, whereas WT decreases were detected later between 8 and 12 months.

“Age-dependent PNN reductions were evident in both genotypes and regions, resulting in even greater PNN loss with disease progression in 5xFAD mice; WFA+ PNNs were significantly reduced by 12mo (p<0.01) and 8mo (p<0.05) in the diseased subiculum and visual cortex, respectively, compared to the same 5xFAD regions at 4mo, whereas significant within-genotype decreases in WT subiculum and cortical PNNs”

May combine two claims.

AD patient brains exhibited significantly fewer PNNs and more Thio-S+ dense-core plaques compared to non-demented control brains, with a highly significant negative correlation between ACAN+ PNNs and Thio-S+ plaques across samples.

“We found that AD patient brains exhibited significantly fewer PNNs (p<0.01; Fig. 3E) and more Thio-S+ dense-core plaques (p<0.001; Fig. 3F) compared to non-demented control brains, such that a highly significant negative correlation between ACAN+ PNNs and Thio-S+ plaques was observed across unaffected and diseased tissue samples (Spearman's r=−0.49, p<0.0001; Fig. 3G).”

• AssumptionRests on an assumption: The human middle frontal gyrus is representative of AD cortical PNN changes.

May combine two claims.

Mean intensity of WFA+ signal colocalizing with subiculum PV+ neurons was significantly reduced at every age compared to WT, indicating that PV+ PNN component density and/or integrity is diminished prior to PV+ neuronal loss and remains impaired in surviving cells.

“Mean intensity of WFA+ signal colocalizing with subiculum PV+ neurons, a measure of the structural integrity of interneuron-associated PNNs, was significantly reduced at every age compared to WT (p<0.01, p<0.0001, p<0.0001, p<0.01 starting at 4mo; Fig. 2C), indicating that PV+ PNN component density and/or integrity is diminished prior to PV+ neuronal loss, and remains impaired in surviving cells”

May combine two claims.

Aggrecan staining at 12 months confirmed morphological abnormalities and loss of 5xFAD subiculum and cortical PNNs.

“Finally, disease-related effects on PNNs were validated at 12mo by staining for aggrecan, a core PNN CSPG [24], which confirmed the morphological abnormalities as well as the loss of 5xFAD subiculum and cortical PNNs at this timepoint (p<0.001, p<0.05; Fig. S2D-F).”

• cites [24] Rowlands, 2018, Aggrecan Directs Extracellular Matrix-Mediated Neuronal Plasticity, J. Neurosci. sourceNot checked yet

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May combine two claims.

Small debris-like WFA+ puncta, distinct from PNNs, were abundant throughout the 5xFAD brain by 18 months, particularly in the hippocampus, where they associated with PAM and NPAM.

“Interestingly, small debris-like WFA+ puncta, phenotypically distinct from PNNs, were abundant throughout the 5xFAD brain by 18mo, particularly in the hippocampus (e.g. subiculum, CA1) where they associated with PAM (Fig. 2D; arrowheads) and NPAM (Fig. S4A; arrowheads).”

May combine two claims.

WFA-positive components are observable within 5xFAD microglia, primarily in plaque-associated microglia, at 18 months.

“(J) Representative 63X image and (K) 3D rendering of WFA+ components observable within 5xFAD microglia (arrowheads; primarily in PAM) at 18 months.”

May combine two claims.

Plaques were evident in multiple brain regions by 4 months in male and female 5xFAD mice, concomitant with activation and gliosis of plaque-associated and non-plaque-associated microglia.

“Plaques were evident in multiple brain regions by 4mo in male and female 5xFAD mice, concomitant with the stereotypical activation and gliosis of plaque- and non-plaque-associated microglia (PAM and NPAM, respectively; Fig. 1A-C) [52, 53].”

• cites [52] Heneka, 2015, Neuroinflammation in Alzheimer's disease, The Lancet Neurology sourceNot checked yet

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• cites [53] Reed-Geaghan, 2020, Plaque-associated myeloid cells derive from resident microglia in an Alzheimer's disease model, Journal of Experimental Medicine sourceNot checked yet

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May combine two claims.

CS-56 analysis revealed significant increases in total extracellular CSPG in the subiculum but not visual cortex of 5xFAD mice at early stages of PNN loss.

“Analysis of total extracellular CSPG with CS-56 at early stages of PNN loss, which generally labels CSPGs excluded from PNNs e.g. those found in glial scars [54, 55], revealed significant increases in the subiculum but not visual cortex (p<0.05, p = 0.0818) of 5xFAD mice (Fig. S2A-C).”

• cites [54] Pearson, 2018, Identification of a critical sulfation in chondroitin that inhibits axonal regeneration, Elife sourceNot checked yet

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• cites [55] Yi, 2012, Alterations in sulfated chondroitin glycosaminoglycans following controlled cortical impact injury in mice, J. Comp. Neurol. sourceNot checked yet

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May combine two claims.

Microglial numbers are significantly increased compared to wild-type at every age in the subiculum and from 8 months on in the visual cortex of 5xFAD mice.

“(D-E) Microglial numbers were significantly increased compared to WT at every age in the subiculum and from 8mo on in the visual cortex (all p<0.0001; 2-way ANOVAs with Tukey's post-hoc test; n = 8–10/group).”

May combine two claims.

Microglia have a homeostatic role in the maintenance of perineuronal nets, such that basal perineuronal net levels in the healthy adult brain are elevated following microglial depletion.

“It is interesting to note that we identified here and elsewhere [36] a homeostatic role for microglia in the maintenance of PNNs, such that basal PNN levels in the healthy adult brain are elevated following microglial depletion.”

• cites [36] Crapser, 2019, Microglial depletion prevents extracellular matrix changes and striatal volume reduction in a model of Huntington's disease, Brain sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

The appearance and spread of Alzheimer's disease pathological substrates is fundamentally linked to a cascade of events that results in synaptic dysfunction and neuronal loss, manifesting behaviorally as progressive impairments in memory and cognition.

“The appearance and spread of these pathological substrates is fundamentally linked to a cascade of events that results in the synaptic dysfunction and neuronal loss characteristic of the disease, manifesting behaviorally as progressive impairments in memory and cognition [1].”

• cites [1] Selkoe, 2016, The amyloid hypothesis of Alzheimer's disease at 25 years, EMBO Mol Med sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Enhanced plasticity and learning is observed following experimental perineuronal net ablation.

“enhanced plasticity and learning is observed following experimental PNN ablation [23, 24]”

• cites [23] Pizzorusso, 2002, Reactivation of Ocular Dominance Plasticity in the Adult Visual Cortex, Science sourceNot checked yet

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• cites [24] Rowlands, 2018, Aggrecan Directs Extracellular Matrix-Mediated Neuronal Plasticity, J. Neurosci. sourceNot checked yet

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May combine two claims.

The majority of Alzheimer's disease risk genes are highly or solely expressed in myeloid cells, implicating microglia in Alzheimer's disease etiology.

“the majority of these risk genes are highly or solely expressed in myeloid cells (e.g. Trem2, Cd33, Spi1, Abi3, Plcg2, Apoe) thereby implicating microglia in AD etiology [4, 5].”

• cites [4] Sierksma, A., Lu, A., Mancuso, R., Fattorelli, N., Thrupp, N., Salta, E., Zoco, J., Blum, D., Buée, L., De Strooper, B., et al. Novel Alzheimer risk genes determine the microglia response to amyloid-β but not to TAU pathology. EMBO Mol Medn/a, e10606.Not checked yet

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• cites [5] Sims, 2017, Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer's disease, Nat. Genet. sourceNot checked yet

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Classified as probably not a substantial claim. May combine two claims.

Perineuronal nets form preferentially around fast-spiking parvalbumin-positive GABAergic interneurons throughout the brain during closure of the critical period of plasticity, locking proximal synapses in place and providing synaptic stability.

“PNNs form preferentially around fast-spiking parvalbumin (PV)+ GABAergic interneurons throughout the brain during the closure of the critical period of plasticity, effectively “locking” proximal synapses in place and providing synaptic stability [21] soon after synaptic pruning is completed by developmental microglia [22].”

• cites [21] Fawcett, 2019, The roles of perineuronal nets and the perinodal extracellular matrix in neuronal function, Nature Reviews Neuroscience sourceNot checked yet

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• cites [22] Paolicelli, 2011, Synaptic Pruning by Microglia Is Necessary for Normal Brain Development, Science sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Genes expressed in other CNS cell types have microglia-specific enhancers with risk polymorphisms that selectively affect myeloid expression.

“genes expressed in other CNS cell types (e.g. neurons and astrocytes) have microglia-specific enhancers with risk polymorphisms that selectively affect myeloid expression (i.e. Bin1) [6].”

• cites [6] Nott, 2019, Brain cell type–specific enhancer–promoter interactome maps and disease<strong>-</strong>risk association, Science sourceNot checked yet

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Classified as probably not a substantial claim. May combine two claims.

Structural modification of perineuronal nets alters synaptic transmission, synapse number, and the ion channel/neurotransmitter receptor composition of synapses.

“the structural modification of PNNs alters synaptic transmission [27], synapse number [28], and the ion channel/neurotransmitter receptor composition of synapses [29]”

• cites [27] Blosa, 2015, The extracellular matrix molecule brevican is an integral component of the machinery mediating fast synaptic transmission at the calyx of Held, J. Physiol. (Lond.) sourceNot checked yet

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• cites [28] Gottschling, 2019, Elimination of the four extracellular matrix molecules tenascin-C, tenascin-R, brevican and neurocan alters the ratio of excitatory and inhibitory synapses, Sci Rep sourceNot checked yet

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• cites [29] Favuzzi, 2017, Activity-Dependent Gating of Parvalbumin Interneuron Function by the Perineuronal Net Protein Brevican, Neuron sourceNot checked yet

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Classified as probably not a substantial claim.

Whether perineuronal nets are altered in Alzheimer's disease remains controversial.

“but whether PNNs are altered in AD remains controversial.”

• Also stated in: § Introduction · plain

  “the extent to which PNN loss occurs in human and animal models of AD – where microglia are inextricably linked to disease pathogenesis – remains controversial [40], [41], [42], [43].”

• cites [40] Baig, 2005, Loss of perineuronal net N-acetylgalactosamine in Alzheimer's disease, Acta Neuropathol. sourceNot checked yet

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• cites [41] Cattaud, 2018, Early disruption of parvalbumin expression and perineuronal nets in the hippocampus of the Tg2576 mouse model of Alzheimer's disease can be rescued by enriched environment, Neurobiol. Aging sourceNot checked yet

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• cites [42] Morawski, 2012, Involvement of perineuronal and perisynaptic extracellular matrix in Alzheimer's disease neuropathology, Brain Pathol. sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Microglia interact with and modulate neuronal and synaptic elements, with direct effects on learning and memory.

“evidence indicates that these cells interact with and modulate neuronal and synaptic elements, with direct effects on learning and memory [14], [15], [16], [17], [18], [19].”

• cites [14] Elmore, 2014, Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain, Neuron sourceNot checked yet

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• cites [15] Elmore, 2018, Replacement of microglia in the aged brain reverses cognitive, synaptic, and neuronal deficits in mice, Aging Cell sourceNot checked yet

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• cites [16] Rice, 2015, Elimination of Microglia Improves Functional Outcomes Following Extensive Neuronal Loss in the Hippocampus, J. Neurosci. sourceNot checked yet

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Classified as probably not a substantial claim.

The microglial response to amyloid plaques and/or tau pathology is increasingly thought to cause or contribute heavily to disease-related neurodegeneration.

“the microglial response to amyloid plaques and/or tau pathology is increasingly thought to cause or contribute heavily to disease-related neurodegeneration [10], [11], [12], [13].”

• cites [10] Krasemann, 2017, The TREM2-APOE Pathway Drives the Transcriptional Phenotype of Dysfunctional Microglia in Neurodegenerative Diseases, Immunity sourceNot checked yet

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• cites [11] Shi, 2019, Microglia drive APOE-dependent neurodegeneration in a tauopathy mouse model, J. Exp. Med. sourceNot checked yet

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• cites [12] Spangenberg, 2019, Sustained microglial depletion with CSF1R inhibitor impairs parenchymal plaque development in an Alzheimer's disease model, Nat Commun sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Newly formed memory traces can interfere with the fidelity and recall of previously learned information, as evident by impaired reconsolidation and recall of remote memories following perineuronal net ablation.

“newly formed memory traces can interfere with the fidelity and recall of previously learned information [20], as evident by impaired reconsolidation and recall of remote memories following PNN ablation [25, 26].”

• cites [20] Reichelt, 2019, Perineuronal Nets: plasticity, Protection, and Therapeutic Potential, Trends Neurosci. sourceNot checked yet

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• cites [25] Shi, 2019, Perineuronal nets protect long-term memory by limiting activity-dependent inhibition from parvalbumin interneurons, Proceedings of the National Academy of Sciences sourceNot checked yet

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• cites [26] Thompson, 2018, Removal of perineuronal nets disrupts recall of a remote fear memory, Proceedings of the National Academy of Sciences sourceNot checked yet

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May combine two claims.

Perineuronal net loss is a salient phenotype of the Alzheimer's disease model and human brain, and a recurring phenotype in the context of neurodegeneration overall.

“Our data suggest that perineuronal net loss is a salient phenotype of the Alzheimer's disease model and human brain, and a recurring phenotype in the context of neurodegeneration overall.”

May combine two claims.

The deleterious effects of AD-associated plaques on PNNs are mediated by microglia, and pre- or post-pathological microglial depletion mitigates PNN loss even without significant changes in plaque load.

“Together, this data collectively suggests that the deleterious effects of AD-associated plaques on PNNs are mediated by microglia, the pre- or post-pathological depletion of which mitigates perineuronal net loss even in the absence of significant changes in plaque load.”

• AssumptionRests on an assumption: The 5xFAD and 3xTg-AD mouse models recapitulate key aspects of human AD pathology relevant to PNN loss and microglial mediation.
• AssumptionRests on an assumption: Microglial depletion with PLX5622 affects PNNs only through microglial loss and not through direct effects on PNNs or other cells.

May combine two claims.

The human PNN-plaque relationship was best described by a nonlinear quadratic model, paralleled by the murine cortex but not subiculum, providing cross-species corroboration on a brain-region dependent relationship between PNNs and plaques.

“Interestingly, these data were best described by a nonlinear quadratic model (Y = 13.91+−5.414*X + 0.7059*X2; r2=0.2094), as paralleled by the distribution of WFA+ PNN area vs. Thio-S+ plaque number in the murine cortex but not subiculum (Fig. S1B,A), providing additional cross-species corroboration on the brain-region dependent relationship between PNNs and plaques.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Microglia release matrix metalloproteinases or other ECM-degrading proteases that act on perineuronal net components in acute injuries such as stroke.

“microglia undergo activation and subsequently release matrix metalloproteinases (e.g. MMP9) or other ECM-degrading proteases that act on PNN components [33].”

• cites [33] Wen, 2018, The Perineuronal ‘Safety’ Net? Perineuronal Net Abnormalities in Neurological Disorders, Front Mol Neurosci sourceNot checked yet

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All correlations reported in the study were confirmed significant using both Pearson's and Spearman's analyses.

“all correlations reported in this study were nonetheless confirmed significant using both analyses”

Classified as probably not a substantial claim.

Dementia and Alzheimer's disease diagnosis were made by a consensus conference using neuropsychological assessment, neurological examination, and medical records following DSM-IV and NINCDS-ADRDA criteria.

“Dementia and AD diagnosis were made by a consensus conference using neuropsychological assessment, neurological examination, and medical records following DSM-IV and National Institute of Neurological and Communicative and Stroke-Alzheimer's Disease and Related Disorders Association criteria, respectively.”

Classified as probably not a substantial claim.

Perineuronal nets protect neurons against tau and amyloid-beta pathology.

“PNNs are reported to protect neurons against tau [37, 38] and Aβ pathology [39]”

• cites [37] Brückner, 1999, Cortical areas abundant in extracellular matrix chondroitin sulphate proteoglycans are less affected by cytoskeletal changes in Alzheimer's disease, Neuroscience sourceNot checked yet

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• cites [38] Morawski, 2010, Neurons associated with aggrecan-based perineuronal nets are protected against tau pathology in subcortical regions in Alzheimer's disease, Neuroscience sourceNot checked yet

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• cites [39] Miyata, 2007, Perineuronal nets protect against amyloid beta-protein neurotoxicity in cultured cortical neurons, Brain Res sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Assignment of animals to treatment groups was random and balanced for sex, and researchers were blinded to genotype and treatment groups during histological analysis.

“Assignment of animals to treatment groups was conducted in a random manner and was balanced for sex, and researchers were blinded to genotype and treatment groups during analysis of histological data.”

Classified as probably not a substantial claim. May combine two claims.

Neuropathological examination included Braak and Braak staging for plaques and tangles and diagnosis of neuropathological Alzheimer's disease using National Institute on Aging-Reagan criteria.

“Neuropathological examination included Braak and Braak staging for plaques and tangles and diagnosis of neuropathological AD using National Institute on Aging-Reagan criteria [47].”

• cites [47] Hyman, 2012, National Institute on Aging-Alzheimer's Association guidelines for the neuropathologic assessment of Alzheimer's disease, Alzheimers Dement sourceNot checked yet

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May combine two claims.

Microglia express and are critically dependent upon signaling through the myeloid CSF1/IL-34 receptor CSF1R for survival.

“Microglia express and are critically dependent upon signaling through the myeloid CSF1/IL-34 receptor CSF1R for survival [14].”

• cites [14] Elmore, 2014, Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain, Neuron sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Microglia-mediated perineuronal net loss occurs in Huntington's disease.

“recent work from our lab identified a role for microglia-mediated PNN loss in Huntington's disease [36].”

• cites [36] Crapser, 2019, Microglial depletion prevents extracellular matrix changes and striatal volume reduction in a model of Huntington's disease, Brain sourceNot checked yet

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May combine two claims.

PNN components were observed within microglia in both mice and humans, suggesting that microglia phagocytose these ECM structures directly or the debris resulting from their degradation, as well as within human dense-core plaques.

“We observed PNN components within microglia in both mice and humans, suggesting that microglia phagocytose these ECM structures directly or the debris resulting from their degradation (e.g. following enzymatic breakdown), as well as within human dense-core plaques.”

May combine two claims.

Microglia are critical homeostatic regulators of PNNs in the healthy adult brain, and their phenotypic changes in response to plaques mediate extensive PNN loss in AD.

“In conclusion, our data show that microglia are critical homeostatic regulators of PNNs in the healthy adult brain, and that their phenotypic changes in response to plaques mediate extensive PNN loss in AD.”

• AssumptionRests on an assumption: The 5xFAD and 3xTg-AD mouse models recapitulate key aspects of human AD pathology relevant to PNN loss and microglial mediation.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

5xFAD mice display synaptic deficits and neuronal loss at later stages of disease.

“in addition to synaptic deficits and neuronal loss at later stages of the disease [49, 45].”

• cites [49] Buskila, 2013, Synaptic deficits in layer 5 neurons precede overt structural decay in 5xFAD mice, Neuroscience sourceNot checked yet

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• cites [45] Jawhar, 2012, Motor deficits, neuron loss, and reduced anxiety coinciding with axonal degeneration and intraneuronal Aβ aggregation in the 5XFAD mouse model of Alzheimer's disease, Neurobiol. Aging sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Microglia from 5xFAD mice show canonical upregulation of disease-associated microglial markers (e.g. Cst7, Axl, Trem2, Apoe, Ctss) and loss of homeostatic markers (P2ry12, Tmem119).

“Microglia from these mice have been extensively characterized, revealing the canonical upregulation of disease-associated microglial markers (e.g. Cst7, Axl, Trem2, Apoe, Ctss) and loss of homoestatic markers (P2ry12, Tmem119) that reflect disease-related changes in form and function [12, 50, 51].”

• cites [12] Spangenberg, 2019, Sustained microglial depletion with CSF1R inhibitor impairs parenchymal plaque development in an Alzheimer's disease model, Nat Commun sourceNot checked yet

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• cites [50] Griciuc, 2019, TREM2 Acts Downstream of CD33 in Modulating Microglial Pathology in Alzheimer's Disease, Neuron sourceNot checked yet

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• cites [51] Keren-Shaul, 2017, A Unique Microglia Type Associated with Restricting Development of Alzheimer's Disease, Cell sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Perineuronal net loss is reported in seizure and prion disease.

“PNN loss is also reported in other neurological disorders such as seizure [34] and prion disease [35]”

• cites [34] Rankin-Gee, 2015, Perineuronal net degradation in epilepsy, Epilepsia sourceNot checked yet

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• cites [35] Franklin, 2008, Loss of Perineuronal Net in ME7 Prion Disease, J. Neuropathol. Exp. Neurol. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

PLX5622 significantly reduced Thio-S+ plaque numbers in the visual cortex but not subiculum, suggesting that microglial depletion can prevent PNN loss even without significant changes in plaque load.

“PLX5622 significantly reduced Thio-S+ plaque numbers in the visual cortex (p<0.001) but not subiculum (p = 0.0749; two-tailed unpaired t-tests; n = 6–9/group), together suggesting that microglial depletion can prevent PNN loss even without significant changes in plaque load.”

May combine two claims.

Neuronal and synaptic genes and related functional pathways were extensively and uniquely downregulated in the 7-month 5xFAD hippocampus compared to WT hippocampus and other 5xFAD brain regions, and subiculum neurons and dendritic spines were lost in 10-month-old 5xFAD mice.

“We previously reported that neuronal and synaptic genes (e.g. Dlk2, Kcnq3, Nrg3, Scn1b) and related functional pathways (e.g. synaptic vesicles, glutamate receptors, neuronal membranes) were extensively and uniquely downregulated in the 7mo 5xFAD hippocampus compared to WT hippocampus and other 5xFAD brain regions [12], respectively, in addition to a loss of subiculum neurons and dendritic spines”

• cites [12] Spangenberg, 2019, Sustained microglial depletion with CSF1R inhibitor impairs parenchymal plaque development in an Alzheimer's disease model, Nat Commun sourceNot checked yet

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• cites [13] Spangenberg, 2016, Eliminating microglia in Alzheimer's mice prevents neuronal loss without modulating amyloid-beta pathology, Brain sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Contrasting effects of PNN ablation on certain memory tasks and measures of plasticity suggest this is not the case for all types of memory, but differences may be partly attributable to the PNN ablation method used.

“Although contrasting effects of PNN ablation on certain (specifically object recognition) memory tasks and measures of plasticity suggest this is not the case for all types of memory [21, 29, 87], such differences may also be at least partly attributable to the PNN ablation method utilized (e.g. enzymatic digestion vs genetic deletion of PNN components) which can produce differential effects on”

• cites [21] Fawcett, 2019, The roles of perineuronal nets and the perinodal extracellular matrix in neuronal function, Nature Reviews Neuroscience sourceNot checked yet

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• cites [29] Favuzzi, 2017, Activity-Dependent Gating of Parvalbumin Interneuron Function by the Perineuronal Net Protein Brevican, Neuron sourceNot checked yet

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• cites [87] Romberg, 2013, Depletion of perineuronal nets enhances recognition memory and long-term depression in the perirhinal cortex, J. Neurosci. sourceNot checked yet

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• cites [82] Bukalo, 2001, Modification of extracellular matrix by enzymatic removal of chondroitin sulfate and by lack of tenascin-R differentially affects several forms of synaptic plasticity in the hippocampus, Neuroscience sourceNot checked yet

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Classified as probably not a substantial claim. May combine two claims.

Genetic ablation of PNN structural components and consequent loss of neuronal PNN integrity can induce loss of synapses and impaired synaptic colocalization/expression of neurotransmitter receptor subunits, potentially manifesting as deficits in synaptic plasticity.

“This may be due in part to the degradation of the PNNs in which synapses are embedded, as the genetic ablation of one or more PNN structural components and consequent loss of neuronal PNN integrity can induce the loss of synapses [28, 80] and impaired synaptic colocalization/expression of neurotransmitter receptor subunits [29], potentially manifesting as deficits in synaptic plasticity (e.g. LTP)”

• cites [28] Gottschling, 2019, Elimination of the four extracellular matrix molecules tenascin-C, tenascin-R, brevican and neurocan alters the ratio of excitatory and inhibitory synapses, Sci Rep sourceNot checked yet

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• cites [80] Geissler, 2013, Primary Hippocampal Neurons, Which Lack Four Crucial Extracellular Matrix Molecules, Display Abnormalities of Synaptic Structure and Function and Severe Deficits in Perineuronal Net Formation, The Journal of Neuroscience sourceNot checked yet

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• cites [29] Favuzzi, 2017, Activity-Dependent Gating of Parvalbumin Interneuron Function by the Perineuronal Net Protein Brevican, Neuron sourceNot checked yet

  Not checked in the free run.

• cites [81], [82], [83]Not checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Synaptic loss is a key feature of AD pathogenesis mediated, at least in part, by microglia.

“For instance, synaptic loss is a key feature of AD pathogenesis mediated, at least in part, by microglia [13, 79].”

• cites [13] Spangenberg, 2016, Eliminating microglia in Alzheimer's mice prevents neuronal loss without modulating amyloid-beta pathology, Brain sourceNot checked yet

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• cites [79] Hong, 2016, Complement and microglia mediate early synapse loss in Alzheimer mouse models, Science sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Enzymatic degradation of auditory cortex PNNs with chondroitinase ABC disrupts consolidation of auditory fear learning, which is restored upon PNN recovery, whereas PNN digestion in the amygdala or visual cortex disrupts recall of contextual fear memories and remote visual fear memories, respectively.

“Of further relevance to AD, the enzymatic degradation of auditory cortex PNNs with chondroitinase ABC (chABC) disrupts consolidation of auditory fear learning [84], which is restored upon PNN recovery, whereas PNN digestion in the amygdala or visual cortex disrupts the recall of contextual fear memories [85] and remote visual fear memories [26], respectively.”

• cites [84] Banerjee, 2017, Perineuronal Nets in the Adult Sensory Cortex Are Necessary for Fear Learning, Neuron sourceNot checked yet

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• cites [85] Gogolla, 2009, Perineuronal Nets Protect Fear Memories from Erasure, Science sourceNot checked yet

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• cites [26] Thompson, 2018, Removal of perineuronal nets disrupts recall of a remote fear memory, Proceedings of the National Academy of Sciences sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

PNNs have been proposed as the molecular substrate underpinning long-term memory storage, by serving as a scaffold that retains synapse position and strength.

“In light of such data, PNNs have been proposed as the molecular substrate underpinning long-term memory storage, by serving as a scaffold that retains synapse position and strength [86].”

• cites [86] Tsien, 2013, Very long-term memories may be stored in the pattern of holes in the perineuronal net, Proceedings of the National Academy of Sciences sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Enhanced plasticity and learning can impair storage and recall of remote memories due to interference from new memory traces, and so enhancement of these factors following PNN loss would still be consistent with impaired reconsolidation and recall of remote, long-term memories.

“On the other hand, enhanced plasticity and learning can impair the storage and recall of remote memories due to interference from new memory traces [20], and so the enhancement of these factors following PNN loss would still be consistent with the consequently impaired reconsolidation and recall of remote, long-term memories reported by others [25, 26].”

• cites [20] Reichelt, 2019, Perineuronal Nets: plasticity, Protection, and Therapeutic Potential, Trends Neurosci. sourceNot checked yet

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• cites [25] Shi, 2019, Perineuronal nets protect long-term memory by limiting activity-dependent inhibition from parvalbumin interneurons, Proceedings of the National Academy of Sciences sourceNot checked yet

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• cites [26] Thompson, 2018, Removal of perineuronal nets disrupts recall of a remote fear memory, Proceedings of the National Academy of Sciences sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Microglial clearance of extracellular matrix in response to neuronal IL-33 promotes synaptic remodeling and plasticity in mice.

“Recent work in mice shows that microglial clearance of ECM in response to neuronal IL-33 promotes synaptic remodeling and plasticity [89].”

• cites [89] Nguyen, 2020, Microglial Remodeling of the Extracellular Matrix Promotes Synapse Plasticity, Cell. sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Microglial depletion prevents the forgetting of remote memories in healthy adult mice.

“Microglial depletion also enhances performance in certain memory tasks [12, 14, 16] and prevents the forgetting of remote memories, and in association, the dissociation of memory engram cells in healthy adult mice [18].”

• cites [18] Wang, 2020, Microglia mediate forgetting via complement-dependent synaptic elimination, Science sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Microglial depletion enhances performance in certain memory tasks in healthy adult mice.

“Microglial depletion also enhances performance in certain memory tasks [12, 14, 16] and prevents the forgetting of remote memories, and in association, the dissociation of memory engram cells in healthy adult mice [18].”

• cites [12] Spangenberg, 2019, Sustained microglial depletion with CSF1R inhibitor impairs parenchymal plaque development in an Alzheimer's disease model, Nat Commun sourceNot checked yet

  Not checked in the free run.

• cites [14] Elmore, 2014, Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain, Neuron sourceNot checked yet

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• cites [16] Rice, 2015, Elimination of Microglia Improves Functional Outcomes Following Extensive Neuronal Loss in the Hippocampus, J. Neurosci. sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Microglial depletion is associated with dissociation of memory engram cells in healthy adult mice.

“Microglial depletion also enhances performance in certain memory tasks [12, 14, 16] and prevents the forgetting of remote memories, and in association, the dissociation of memory engram cells in healthy adult mice [18].”

• cites [18] Wang, 2020, Microglia mediate forgetting via complement-dependent synaptic elimination, Science sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Perineuronal net digestion enhances learning rate.

“Supporting this, a recent study found that while PNN digestion enhanced learning rate, it ultimately impaired memory retention, due in part to the extensive synaptic remodeling induced by PNN loss [88].”

• cites [88] Carulli, 2020, Cerebellar plasticity and associative memories are controlled by perineuronal netsNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

PNN formation in the hippocampus is required for consolidation and reconsolidation of recent and remote fear memories.

“Similarly, PNN formation in the hippocampus is required for the consolidation and reconsolidation of recent and remote fear memories [25].”

• cites [25] Shi, 2019, Perineuronal nets protect long-term memory by limiting activity-dependent inhibition from parvalbumin interneurons, Proceedings of the National Academy of Sciences sourceNot checked yet

  Not checked in the free run.