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How abnormal neurodevelopment relates to the tumour aggressiveness of medulloblastoma (MB), the most common type of embryonal tumour, remains elusive. Here we uncover a neurodevelopmental epigenomic programme that is hijacked to induce MB metastatic dissemination. Unsupervised analyses of integrated publicly available datasets with our newly generated data reveal that SMARCD3 (also known as BAF60C) regulates Disabled 1 (DAB1)-mediated Reelin signalling in Purkinje cell migration and MB metastasis by orchestrating cis-regulatory elements at the DAB1 locus. We further identify that a core set of transcription factors, enhancer of zeste homologue 2 (EZH2) and nuclear factor I X (NFIX), coordinates with the cis-regulatory elements at the SMARCD3 locus to form a chromatin hub to control SMARCD3 expression in the developing cerebellum and in metastatic MB. Increased SMARCD3 expression activates Reelin–DAB1-mediated Src kinase signalling, which results in a MB response to Src inhibition. These data deepen our understanding of how neurodevelopmental programming influences disease progression and provide a potential therapeutic option for patients with MB.

The amyloid precursor protein (APP) is linked to the genetics and pathogenesis of Alzheimer's disease (AD). It is the parent protein of the β-amyloid (Aβ) peptide, the main constituent of the amyloid plaques found in an AD brain. The pathways from APP to Aβ are intensively studied, yet the normal functions of APP itself have generated less interest. We report here that glutamate stimulation of neuronal activity leads to a rapid increase inAppgene expression. In mouse and human neurons, elevated APP protein changes the structure of the axon initial segment (AIS) where action potentials are initiated. The AIS is shortened in length and shifts away from the cell body. The GCaMP8f Ca2+reporter confirms the predicted decrease in neuronal activity. NMDA antagonists or knockdown ofAppblock the glutamate effects. The actions of APP on the AIS are cell-autonomous; exogenous Aβ, either fibrillar or oligomeric, has no effect. In culture, APPSwe(a familial AD mutation) induces larger AIS changes than wild type APP. Ankyrin G and βIV-spectrin, scaffolding proteins of the AIS, both physically associate with APP, more so in AD brains. Finally, in humans with sporadic AD or in the R1.40 AD mouse model, both females and males, neurons have elevated levels of APP protein that invade the AIS.In vivoasin vitro, this increased APP is associated with a significant shortening of the AIS. The findings outline a new role for the APP and encourage a reconsideration of its relationship to AD.SIGNIFICANCE STATEMENTWhile the amyloid precursor protein (APP) has long been associated with Alzheimer's disease (AD), the normal functions of the full-length Type I membrane protein have been largely unexplored. We report here that the levels of APP protein increase with neuronal activity.In vivoandin vitro, modest amounts of excess APP alter the properties of the axon initial segment. The β-amyloid peptide derived from APP is without effect. Consistent with the observed changes in the axon initial segment which would be expected to decrease action potential firing, we show that APP expression depresses neuronal activity. In mouse AD models and human sporadic AD, APP physically associates with the scaffolding proteins of the axon initial segment, suggesting a relationship with AD dementia.

Adult-onset neurodegenerative diseases are often accompanied by evidence of a chronic inflammation that includes activation of microglial cells and altered levels of brain cytokines. Aspects of this response are likely secondary reactions to neurodegeneration, but for many illnesses the inflammation may itself be an early and even causative disease event. In such cases, the inflammation is referred to as “sterile” as it occurs in the absence of an actual bacterial or viral pathogen. A potent trigger of sterile inflammation in CNS microglia has been shown to be the presence of DNA in the cytoplasm (cytoDNA) induced either by direct DNA damage or by inhibited DNA repair. We have shown that cytoDNA comes from the cell nucleus as a result of insufficient DNA damage repair. Using wild-type andAtm-/-mouse microglia, we extend these observations here by showing that its genomic origins are not random, but rather are heavily biased toward transcriptionally inactive, intergenic regions, in particular repetitive elements and AT-rich sequences. Once released from the genome, in both males and females, we show that cytoDNA is actively exported to the cytoplasm by a CRM1-dependent mechanism. In the cytoplasm, it is degraded either by a cytosolic exonuclease, Trex1, or an autophagy pathway that ends with degradation in the lysosome. Blocking the accumulation of cytoDNA prevents the emergence of the sterile inflammation reaction. These findings offer new insights into the emergence of sterile inflammation and offer novel approaches that may be of use in combatting a wide range of neurodegenerative conditions.SIGNIFICANCE STATEMENTSterile inflammation describes a state where the defenses of the immune system are activated in the absence of a true pathogen. A potent trigger of this unorthodox response is the presence of DNA in the cytoplasm, which immune cells interpret as an invading virus or pathogen. We show that when DNA damage increases, fragments of the cell's own genome are actively exported to the cytoplasm where they are normally degraded. If this degradation is incomplete an immune reaction is triggered. Both age and stress increase DNA damage, and as age-related neurodegenerative diseases are frequently accompanied by a chronic low-level inflammation, strategies that reduce the induction of cytoplasmic DNA or speed its clearance become attractive therapeutic targets.

Carriers of the APOE4 (Apolipoprotein E ε4) variant of the APOE gene are subject to several age-related health risks, including Alzheimer's disease (AD). The deficient lipid and cholesterol transport capabilities of the APOE4 protein are one reason for the altered risk profile. In particular, APOE4 carriers are at elevated risk for sporadic Alzheimer's disease (AD). While deposits o misfolded proteins are present in the AD brain, white matter (WM) myelin is also disturbed. As myelin is a lipid- and cholesterol-rich structure, the connection to APOE makes considerable biological sense. To explore the APOE-WM connection, we have analyzed the impact of human APOE4 on oligodendrocytes (OLs) of the mouse both in vivo and in vitro. We find that APOE proteins is enriched in astrocytes but sparse in OL. In human APOE4 (hAPOE4) knockin mice, myelin lipid content is increased but the density of major myelin proteins (MBP, MAG and PLP) is largely unchanged. We also find an unexpected but significant reduction of cell density of the OL lineage (Olig2

Given the past two decades of over 40 failed trials of amyloid-lowering therapies in Alzheimer's Disease (AD), many of which succeeded in lowering amyloid as designed, we present an ethical argument for emptying the drug pipeline of tests of amyloid-lowering agents so as to end the historical dominance of the amyloid-reducing therapeutic approach in AD.

The glutamatergic cycle is essential in modulating memory processing by the hippocampal circuitry. Our combined proton magnetic resonance spectroscopy (1 H-MRS) and task-based functional magnetic resonance imaging (fMRI) study (using face-name paired-associates encoding and retrieval task) of a cognitively normal cohort of 67 healthy adults (18 ApoE4 carriers and 49 non-ApoE4 carriers) found altered patterns of relationships between glutamatergic-modulated synaptic signalling and neuronal activity or functional hyperaemia in the ApoE4 isoforms. Our study highlighted the asymmetric left-right hippocampal glutamatergic system in modulating neuronal activities in ApoE4 carriers versus non-carriers. Such brain differentiation might be developmental cognitive advantages or compensatory due to impaired synaptic integrity and plasticity in ApoE4 carriers. As there was no difference in myoinositol levels measured by MRS between the ApoE4 and non-ApoE4 subgroups, the mechanism is unlikely to be a response to neuroinflammation.

White matter degradation in the frontal lobe is one of the earliest detectable changes in aging and Alzheimer disease. The ε4 allele of apolipoprotein E (APOE4) is strongly associated with such myelin pathology but the underlying cellular mechanisms remain obscure. We hypothesized that, as a lipid transporter, APOE4 directly triggers pathology in the cholesterol-rich myelin sheath independent of AD pathology. To test this, we performed immunohistochemistry on brain tissues from healthy controls, sporadic, and familial Alzheimer disease subjects. While myelin basic protein expression was largely unchanged, in frontal cortex the number of oligodendrocytes (OLs) was significantly reduced in APOE4 brains independent of their Braak stage or NIA-RI criteria. This high vulnerability of OLs was confirmed in humanized APOE3 or APOE4 transgenic mice. A gradual decline of OL numbers was found in the aging brain without associated neuronal loss. Importantly, the application of lipidated human APOE4, but not APOE3, proteins significantly reduced the formation of myelinating OL in primary cell culture derived from Apoe-knockout mice, especially in cholesterol-depleted conditions. Our findings suggest that the disruption of myelination in APOE4 carriers may represent a direct OL pathology, rather than an indirect consequence of amyloid plaque formation or neuronal loss.

Background Apolipoprotein E ɛ4 allele (ApoE4) is the most common gene polymorphism related to Alzheimer's disease (AD). Impaired synaptic dysfunction occurs in ApoE4 carriers before any clinical symptoms. It remains unknown whether ApoE4 status affects the hippocampal neuromodulation, which further influences brain network topology. Purpose To study the relationship of regional and global network properties by using graph theory analysis and glutamatergic (Glx) neuromodulation in the ApoE isoforms. Study Type Prospective. Subjects Eighty‐four cognitively normal adults (26 ApoE4 and 58 non‐ApoE4 carriers). Field Strength/Sequence Gradient‐echo echo‐planar and point resolved spectroscopy sequence at 3 T. Assessment Glx concentration in bilateral hippocampi were processed with jMRUI (4.0), and graph theory metrics (global: γ, λ, small‐worldness in whole brain; regional: nodal clustering coefficient (C i) and nodal characteristic path length (L i)) in top 20% highly connected hubs of subgroups (low‐risk: non‐ApoE4; high‐risk: APOE4) were calculated and compared. Statistical Tests Two‐sample t test was used to compare metrics between subgroups. Correlations between regional properties and Glx by Pearson's partial correlation with false discovery rate correction. Results Significant differences (P

5-hydroxymethylcytosine (5hmC) is an intermediate stage of DNA de-methylation. Its location in the genome also serves as an important regulatory signal for many biological processes and its levels change significantly with the etiology of Alzheimer's disease (AD). In keeping with this relationship, the TET family of enzymes which convert 5-methylcytosine (5mC) to 5hmC are responsive to the presence of Aβ. Using hMeDIP-seq, we show that there is a genome-wide reduction of 5hmC that is found in neurons but not in astrocytes from 3xTg mice (an AD mouse model). Decreased TET enzymatic activities in the brains of persons who died with AD suggest that this reduction is the main cause for the loss of 5hmC. Overexpression of human TET catalytic domains (hTETCDs) from the TET family members, especially for hTET3CD, significantly attenuates the neurodegenerative process, including reduced Aβ accumulation as well as tau hyperphosphorylation, and improve synaptic dysfunction in 3xTg mouse brain. Our findings define a crucial role of deregulated 5hmC epigenetics in the events leading to AD neurodegeneration.

Aim: To explore people’s visual attention and psychological and physiological responses to viewing a Japanese garden (an asymmetrically designed garden) and an herb garden (a symmetrically designed garden). Background: There are few studies of eye movements when observing different style gardens, and how they are connected to the interpretation of the space, and physiological and psychological responses. Method: Thirty subjects were recruited and their physiological and psychological responses to viewing the garden types were assessed using a heart-rate monitor and questionnaire. Eye movements while viewing projected slide images of the gardens were tracking using an eye-tracking monitor. Results: A significant decrease in heart rate was observed when subjects were viewing the Japanese garden as opposed to viewing the herb garden. Mood was significantly improved in both gardens, but eye-gaze patterns differed. The Japanese garden elicited far more comments about expectations for the coming season; unlike the herb garden, it also induced memories of viewing other landscapes. Conclusion: The physiological and psychological responses to viewing gardens differs based on the quality of landscape design and the prior experience of viewers.

Loss of mitochondrial homeostasis are evident in Alzheimer's disease (AD)1. However, the underlying mechanisms remain largely elusive. In this study, we report that lncMtDloop, an age-related and conserved long noncoding RNA derived from mitochondrial DNA (mtDNA) D-loop, is crucial for sustaining mitochondrial transcription and homeostasis, and associates with Alzheimer’s pathogenesis. Notably, the level of lncMtDloop expression is diminished in the brains of human AD patients and the 3xTg mouse model, whereas the failure to recruit mitochondrial transcription factor A (TFAM) leads to the deregulated mitochondrial transcription. Restoring lncMtDloop expression not only significantly improves mitochondrial transcription, morphology and function, but also enhances mitophagy, ameliorates AD-like pathology, which these subcellular effects extend to a dramatic reversal of deficits in synaptic and cognitive behaviors of the 3xTg mouse model. Collectively, our findings show the decreased lncMtDloop expression potentiates AD risk, thereby revealing an unexpected mitochondrial mechanism contributing to AD pathogenesis and opens a new door for therapeutic intervention.

How dysregulation of neurodevelopment relates to medulloblastoma (MB), the most common pediatric brain tumor, remains elusive. Here, we uncovered a neurodevelopmental epigenomic program being hijacked to induce MB metastatic dissemination. Unsupervised analyses by integrating publicly available datasets with our newly generated data revealed that SMARCD3/BAF60C regulates DAB1-mediated Reelin signaling in Purkinje cell migration and MB metastasis by orchestrating cis-regulatory elements (CREs) at the DAB1 locus. We further identified that a core set of transcription factors, enhancer of zeste homolog 2 (EZH2) and nuclear factor I X (NFIX), coordinates with the CREs at the SMARCD3 locus to form a chromatin hub for controlling SMARCD3 expression in the developing cerebellum and metastatic MB. Elevated SMARCD3 activates Reelin/DAB1-mediated Src kinase signaling, resulting in MB response to Src inhibition. These data deepen our understanding of how neurodevelopmental programming influences disease progression and provide a potential therapeutic option for MB patients.

The small Aβ peptide has been hypothesized to be the main driver of Alzheimer’s disease (AD). Aβ is a proteolytic cleavage product of a larger protein, the amyloid precursor protein (APP), whose normal functions remain largely unexplored. We report here activities of the full-length APP protein that relate directly to the etiology of AD. Increasing neuronal activity leads to a rapid increase in App gene expression. In both cultures of mouse cortical neurons and human iPSC-derived neurons, elevated APP protein changes the structure of the axon initial segment (AIS), the site of action potential initiation. In neurons with elevated APP, the AIS shortens in length and shifts in position away from the cell body. Both changes would be expected to reduce neuronal excitability. The AIS effects are due to the cell-autonomous actions of APP; exogenous Aβ – either fibrillar or oligomeric – has no effect. The findings relate directly to AD in several ways. In culture, APP carrying the Swedish familial AD mutation (APPSwe) induces stronger AIS changes than wild type APP. Ankyrin G and βIV-spectrin, scaffolding proteins of the AIS, both physically associate with APP, and APPSwe binds more avidly than wild type APP. Finally, neurons in the frontal cortex of humans with sporadic AD reveal histologically elevated levels of APP protein that invade the domain of the AIS, whose length is significantly shorter than that found in healthy control neurons. The findings offer an alternative explanation for the effects of at least some familial AD mutations.SignificanceIn familial Alzheimer’s disease (AD) the linkage between the genetics of APP, the neuropathology of the amyloid plaques and the symptoms of dementia are one of the strongest pieces of evidence supporting the amyloid cascade hypothesis – a conceptualization that marks the Aβ peptide as the root cause of AD. Yet, formally, the genetics only point to APP, not its Aβ breakdown product. We report here that the full-length APP protein affects the properties of the axon initial segment and through these changes serves as a dynamic regulator of neuronal activity. We propose that this newly discovered APP function offers a different, Aβ-independent, view of the genetic evidence.

Gene expression studies using chimeric xenograft transplants or co-culture systems have proven to be valuable to uncover cellular dynamics and interactions during development or in disease models. However, the mRNA sequence similarities among species presents a challenge for accurate transcript quantification. To identify optimal strategies for analyzing mixed-species RNA sequencing data, we evaluate both alignment-dependent and alignment-independent methods. Alignment of reads to a pooled reference index is effective, particularly if optimal alignments are used to classify sequencing reads by species, which are re-aligned with individual genomes, generating >97% accuracy across a range of species ratios. Alignment-independent methods, such as Convolutional Neural Networks, which extract the conserved patterns of sequences from two species, classify RNA sequencing reads with over 85% accuracy. Importantly, both methods perform well with different ratios of human and mouse reads. Our evaluation identifies valuable and effective strategies to dissect species composition of RNA sequencing data from mixed populations.

Alzheimer's disease (AD) is a prevalent, progressive and irreversible, neurodegenerative disease with no disease modifying treatment yet available. The projected burden of AD on our healthcare system is immense and thus there is an immediate need for new drugs that prevent or attenuate AD symptoms. While most efforts in the field are directed at treatments that reduce amyloid or tau burden in the brain, we have taken an alternate approach - a model based on reducing AD-associated neuronal cell cycle events. Using this model, we have screened a largely unexplored source of compounds with therapeutic potential - the natural products created by diverse strains of marine bacteria. Two hundred and twenty-five bacterial extracts from different strains were tested for both toxicity and neuroprotective properties by crystal violet and In-cell Western - first in HT22 cells and then in mouse primary neuronal cultures. Based on these screens, we have identified several promising leads, and here we focus on the most promising of these. We found that we could directly assay even a crude bacterial extract in our E16 mouse cortical neuronal cultures and screen for activities that prevent cell cycle reentry and preserve synaptic structure. Preliminary tests in 1-month-old animals from a mouse model of Ataxia telangiectasia, showed that blockage of cell cycle-related neuronal death could also be successful in vivo. This adds an important extension to our in vitro studies. These findings showcase a new effective and efficient assay system and validate the use of marine natural compounds as a novel source for new drugs to fight Alzheimer's disease. Cover Image for this issue: doi: 10.1111/jnc.14733.

This is a pilot study that examines how a person's gaze responds during the observation of a Japanese garden. Thirty-four participants were asked to viewed the same garden in three different ways: directly, through a pane of glass, or as a projected slide. We tracked their eye-movement during the first minutes of observation to observe different gaze strategies used in the different modalities of viewing. Our measurements show that when viewing the garden directly, participants focused on particular elements in their visual field. By contrast, when they viewed a slide they tended to scan the scene more and focus on all elements of the garden evenly. When viewing the real garden through a glass door, the responses were intermediate. Our results demonstrate that the visual evaluation of a space is different depending on the way in which the visual information is presented.

Ataxia-telangiectasia (A-T) is a rare genetic disease caused by mutations in the gene encoding the ATM (Ataxia-telangiectasia mutated) protein. Although neuronal degeneration in the cerebellum remains the most prominent sign in A-T, neuroimaging studies reveal myelin abnormalities as early comorbidities. We hypothesize that these myelin defects are the direct consequence of ATM deficiencies in the oligodendrocytes (OL) lineage. We examined samples from ten A-T brains in which the ATM mutations had been mapped by targeted genomic sequencing as well as samples from Atm-/- mice. In healthy human and wild type mouse cerebellum we confirmed the presence of ATM in white matter OLs. In A-T but not age-matched controls, a significant reduction in OL density was coupled with a massive astrogliosis. We found that the extent of this OL pathology was particularly strong in cases with frameshifts or premature termination ATM mutations. Similar pathologies were also found in Atm-/- mice in an age- and gene dose-dependent fashion. In vitro, DNA damage induced by etoposide-induced DSBs or blockade of ATM activity with KU-60019 differentially jeopardized cell cycle control in OL progenitors and mature OLs. Turning to structural analysis in silico, we identified likely interactions between ATM and myelin basic protein as well as myelin regulatory factor and confirm this by immunoprecipitation. These novel OL-specific functions of the ATM protein affect all stages of the OL lineage. They thus provide a cell biological basis for a direct role for ATM in central nervous system myelination and illustrate how the myelin pathology found in A-T is at least in part independent of neuronal degeneration.

Experiments in primary culture have helped advance our understanding of the curious phenomenon of cell cycle-related neuronal death. In a differentiated postmitotic cell such as a neuron, aberrant cell cycle re-entry is strongly associated with apoptosis. Indeed, in many pathological conditions, neuronal populations at risk for death are marked by cells engaged in a cell cycle like process. The evidence for this conclusion is typically based on finding MAP2-positive cells that are also positive for cell cycle-related proteins (e.g., cyclin D) or have incorporated thymidine analogs such as BrdU or EdU into their nuclei. We now report that we and others may have partly been led astray in pursuing this line of work. Morphometric analysis of mouse embryonic cortical cultures reveals that the size of the 'cycling' MAP2-positive cells is significantly smaller than those of normal neurons, and their expression of MAP2 is significantly lower. This led us to ask whether, rather than representing fully developed neurons, they more closely resembled precursor-like cells. In support of this idea, we find that these small MAP2-positive cells are immunopositive for nestin, a neuronal precursor marker, Olig2, an oligodendrocyte lineage marker, and NG2, an oligodendrocyte precursor marker. Tracking their behavior in culture, we find that they predominantly give rise to GFAP+ astrocytes instead of neurons or oligodendrocytes. These findings argue for a critical reexamination of previous reports of stimuli that lead to neuronal cell cycle-related death in primary cultures.Significance While many laboratories use cultures of rodent embryonic cortex to study the cell biology of brain function, few attend to the identity of the cells in their cultures. We find that a subpopulation of small MAP2-weakly-positive cells, presumed for decades to be neurons, are actually NG2+Olig2+Nestin+ precursor cells that give rise over time mostly to astrocytes. Subjecting the cultures to a challenge meant to mimic an in vivo cell cycle-related neuronal death, we find the percentage of 'cycling' MAP2+ cells does indeed increase, but only because large MAP2+ cells (true neurons) die with no change in their mitotic activity. The MAP2+ precursors remain constant in number and in cell cycle activity. This argues for re-interpretation of experimental neuronal culture data.

ATM (ataxia telangiectasia mutated) protein is found associated with multiple organelles including synaptic vesicles, endosomes and lysosomes, often in cooperation with ATR (ataxia telangiectasia and Rad3 related). Mutation of the ATM gene results in ataxia-telangiectasia (A-T), an autosomal recessive disorder with defects in multiple organs including the nervous system. Precisely how ATM deficiency leads to the complex phenotypes of A-T, however, remains elusive. Here, we reported that part of the connection may lie in autophagy and lysosomal abnormalities. We found that ATM was degraded through the autophagy pathway, while ATR was processed by the proteasome. Autophagy and lysosomal trafficking were both abnormal in atm(−/−) neurons and the deficits impacted cellular functions such as synapse maintenance, neuronal survival and glucose uptake. Upregulated autophagic flux was observed in atm(−/−) lysosomes, associated with a more acidic pH. Significantly, we found that the ATP6V1A (ATPase, H+ transporting, lysosomal V1 subunit A) proton pump was an ATM kinase target. In atm(−/−) neurons, lysosomes showed enhanced retrograde transport and accumulated in the perinuclear regions. We attributed this change to an unexpected physical interaction between ATM and the retrograde transport motor protein, dynein. As a consequence, SLC2A4/GLUT4 (solute carrier family 4 [facilitated glucose transporter], member 4) translocation to the plasma membrane was inhibited and trafficking to the lysosomes was increased, leading to impaired glucose uptake capacity. Together, these data underscored the involvement of ATM in a variety of neuronal vesicular trafficking processes, offering new and therapeutically useful insights into the pathogenesis of A-T. Abbreviations: 3-MA: 3-methyladenine; A-T: ataxia-telangiectasia; ALG2: asparagine-linked glycosylation 2 (alpha-1,3-mannosyltransferase); AMPK: adenosine 5‘-monophosphate (AMP)-activated protein kinase; ATG5: autophagy related 5; ATM: ataxia telangiectasia mutated; ATP6V1A: ATPase, H+ transporting, lysosomal V1 subunit A; ATR: ataxia-telangiectasia and Rad3 related; BFA1: bafilomycin A(1); CC3: cleaved-CASP3; CGN: cerebellar granule neuron; CLQ: chloroquine; CN: neocortical neuron; CTSB: cathepsin B; CTSD: cathepsin D; DYNLL1: the light chain1 of dynein; EIF4EBP1/4E-BP1: eukaryotic translation initiation factor 4E binding protein 1; Etop: etoposide; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HBS: HEPES-buffered saline; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HOMER1: homer protein homolog 1; KU: KU-60019; LAMP1: lysosomal-associated membrane protein 1; LC3B-II: LC3-phosphatidylethanolamine conjugate; Lyso: lysosome; LysopH-GFP: lysopHluorin-GFP; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAP2: microtubule associated protein 2; MAPK14: mitogen-activated protein kinase 14; MAPK8/JNK1: mitogen-activated protein kinase 8; MCOLN1/TRPML1: mucolipin 1; OSBPL1A: oxysterol binding protein like 1A; PIKK: phosphatidylinositol 3 kinase related kinase; Rapa: rapamycin; RILP: rab interacting lysosomal protein; ROS: reactive oxygen species; SEM: standard error of mean; SLC2A4/GLUT4: solute carrier family 2 (facilitated glucose transporter), member 4; TSC2/tuberin: TSC complex subunit 2; ULK1: unc-51 like kinase 1; UPS: ubiquitin-proteasome system; VE: VE-822; WCL: whole-cell lysate; WT: wild type.

Previous research has shown that exposure to Japanese gardens reduces physiological measures of stress, e.g. heart rate, in both healthy subjects and dementia patients. However, the correlation between subjects’ physiological responses and their visual behavior while viewing the garden has not yet been investigated. To address this, we developed a system to collect simultaneous measurements of eye gaze and three physiological indicators of autonomic nervous system activity: electrocardiogram, blood volume pulse, and galvanic skin response. We recorded healthy subjects’ physiological/behavioral responses when they viewed two environments (an empty courtyard and a Japanese garden) in two ways (directly or as a projected 2D photograph). Similar to past work, we found that differences in subject’s physiological responses to the two environments when viewed directly, but not as a photograph. We also found differences in their behavioral responses. We quantified subject’s behavioral responses using several gaze metrics commonly considered to be measures of engagement of focus: average fixation duration, saccade amplitude, spatial entropy and gaze transition entropy. We found decrease in gaze transition entropy, the only metric that accounts for both the spatial and temporal properties of gaze, to have a weak positive correlation with decrease in heart rate. This suggests a relationship between engagement/focus and relaxation. Finally, we found gender differences: females’ gaze patterns were more spatially distributed and had higher transition entropy than males.

Many have criticized the amyloid cascade hypothesis of Alzheimer's disease for its inconsistencies and failures to either accurately predict disease symptoms or guide the development of productive therapies. In addition to criticisms, however, we believe that the field would benefit from having alternative narratives and disease models that can either replace or function alongside of an amyloid-centric view of Alzheimer's. This review is an attempt to meet that need. We offer three experimentally verified amyloid-independent mechanisms, each of which plausibly contributes substantially to the aetiology of Alzheimer's disease: loss of DNA integrity, faulty cell cycle regulation, regression of myelination. We outline the ways in which the failure of each can contribute to AD initiation and progression, and review how, acting alone or in combination with each other, they are sufficient for explaining the full range of AD pathologies. Yet, these three alternatives represent only a few of the many non-amyloid mechanisms that can explain AD pathogenesis. Therefore instead of proposing a single 'alternative hypothesis' to the amyloid cascade theory, sporadic AD is pictured as the result of independent yet intersecting age-related pathologies that afflict the ageing human brain. This article is part of the series "Beyond Amyloid". Cover Image for this issue: doi. 10.1111/jnc.13823.

Myelination is a recent evolutionary addition that significantly enhances the speed of transmission in the neural network. Even slight defects in myelin integrity impair performance and enhance the risk of neurological disorders. Indeed, myelin degeneration is an early and well-recognized neuropathology that is age associated, but appears before cognitive decline. Myelin is only formed by fully differentiated oligodendrocytes, but the entire oligodendrocyte lineage are clear targets of the altered chemistry of the aging brain. As in neurons, unrepaired DNA damage accumulates in the postmitotic oligodendrocyte genome during normal aging, and indeed may be one of the upstream causes of cellular aging - a fact well illustrated by myelin co-morbidity in premature aging syndromes arising from deficits in DNA repair enzymes. The clinical and experimental evidence from Alzheimer's disease, progeroid syndromes, ataxia-telangiectasia and other conditions strongly suggest that oligodendrocytes may in fact be uniquely vulnerable to oxidative DNA damage. If this damage remains unrepaired, as is increasingly true in the aging brain, myelin gene transcription and oligodendrocyte differentiation is impaired. Delineating the relationships between early myelin loss and DNA damage in brain aging will offer an additional dimension outside the neurocentric view of neurodegenerative disease.

Purpose: This study is to examine how viewing a Japanese garden affects Japanese patients with dementia. Background: In a previous study, authors explored the effect on individuals with Alzheimer’s disease of viewing an indoor Japanese garden at a nursing home in the United States and reported that viewing the garden significantly reduced the heart rate, evoked short-term and long-term memories, and improved behavioral symptoms. However, it was unclear whether these effects were caused by the design of Japanese garden or unfamiliarity of the design to Caucasians. Methods: We constructed a Japanese garden on the rooftop of a hospital in Japan and assessed with a total of 25 subjects on the following categories: (1) eye movement, (2) heart rate, and (3) behavior under four different conditions: (a) open view of the site before construction of the Japanese garden (the control space), (b) open view of the Japanese garden, (c) view of the Japanese garden through closed door, and (d) view of Japanese garden through closed door with the chrysanthemum scent. Findings/Results: Viewers’ eyes scanned larger area while viewing the Japanese garden, and viewing the Japanese garden significantly reduced heart rate and improved behavioral symptoms than the control space. We also found that the effect of viewing the same Japanese garden differed across three conditions: the view through an open door, a closed door, and a closed door with added scent.

DNA damage has been reported to induce cell cycle-related neuronal death. This is significant as aberrant cell cycle re-entry of mature, post-mitotic neurons contributes to neurodegeneration. In this study, we investigate how DNA damage elicited by exposure to the topoisomerase I inhibitor camptothecin (CPT) leads to cycle-related death of cultured cortical neurons and examine the function of E2F1 in this process. CPT treatment induced cell cycle initiation of cortical neurons and elevated the expression of certain cell cycle components (e.g., cyclin D1, CDK4, E2F1) but failed to drive S phase entry or DNA synthesis. The arrest in the cell cycle is explained by the elevated expression of the CDK inhibitor p21Cip1. Though its level was increased after CPT treatment, E2F1 did not drive treated neurons into the G1-S phase transition. E2F1 overexpression led to cell cycle activation and acute neuronal apoptosis without detectable entry of the neurons into S phase. ChIPseq analysis demonstrated that E2F1 predominantly occupies positions on or near the promoters of cell cycle related genes. Instead, in CPT-treated neurons, E2F1 preferentially regulated DNA repair related genes. Our study reveals that the functions of E2F1 in postmitotic neurons are context-dependent and offers novel insights into the role of E2F1 in DNA damage induced cycle-related neuronal death.

ATM (ataxia-telangiectasia mutated) is a PI3K-like kinase best known for its role in the DNA damage response (DDR), especially after double-strand breaks. Mutations in the ATM gene result in a condition known as ataxia-telangiectasia (A-T) that is characterized by cancer predisposition, radiosensitivity, neurodegeneration, sterility, and acquired immune deficiency. We show here that the innate immune system is not spared in A-T. ATM-deficient microglia adopt an active phenotype that includes the overproduction of proinflammatory cytokines that are toxic to cultured neurons and likely contribute to A-T neurodegeneration. Causatively, ATM dysfunction results in the accumulation of DNA in the cytoplasm of microglia as well as a variety of other cell types. In microglia, cytoplasmic DNA primes an antiviral response via the DNA sensor, STING (stimulator of interferon genes). The importance of this response pathway is supported by our finding that inhibition of STING blocks the overproduction of neurotoxic cytokines. Cytosolic DNA also activates the AIM2 (absent in melanoma 2) containing inflammasome and induces proteolytic processing of cytokine precursors such as pro-IL-1β. Our study furthers our understanding of neurodegeneration in A-T and highlights the role of cytosolic DNA in the innate immune response. SIGNIFICANCE STATEMENT Conventionally, the immune deficiencies found in ataxia-telangiectasia (A-T) patients are viewed as defects of the B and T cells of the acquired immune system. In this study, we demonstrate the microglia of the innate immune system are also affected and uncover the mechanism by which this occurs. Loss of ATM (ataxia-telangiectasia mutated) activity leads to a slowing of DNA repair and an accumulation of cytoplasmic fragments of genomic DNA. This ectopic DNA induces the antivirus response, which triggers the production of neurotoxic cytokines. This expands our understanding of the neurodegeneration found in A-T and offers potentially new therapeutic options.

Prediabetes and Alzheimer's disease both increase in prevalence with age. The former is a risk factor for the latter, but a mechanistic linkage between them remains elusive. We show that prediabetic serum hyperinsulinemia is reflected in the cerebrospinal fluid and that this chronically elevated insulin renders neurons resistant to insulin. This leads to abnormal electrophysiological activity and other defects. In addition, neuronal insulin resistance reduces hexokinase 2, thus impairing glycolysis. This hampers the ubiquitination and degradation of p35, favoring its cleavage to p25, which hyperactivates CDK5 and interferes with the GSK3β-induced degradation of β-catenin. CDK5 contributes to neuronal cell death while β-catenin enters the neuronal nucleus and re-activates the cell cycle machinery. Unable to successfully divide, the neuron instead enters a senescent-like state. These findings offer a direct connection between peripheral hyperinsulinemia, as found in prediabetes, age-related neurodegeneration and cognitive decline. The implications for neurodegenerative conditions such as Alzheimer's disease are described.

Background Synaptic protein dyshomeostasis and functional loss is an early invariant feature of Alzheimer’s disease (AD), yet the unifying etiological pathway remains largely unknown. Knowing that cyclin-dependent kinase 5 (CDK5) plays critical roles in synaptic formation and degeneration, its phosphorylation targets were reexamined in search of candidates with direct global impacts on synaptic protein dynamics, and the associated regulatory network was also analyzed. Methods Quantitative phosphoproteomics and bioinformatics analyses were performed to identify top-ranked candidates. A series of biochemical assays was used to investigate the associated regulatory signaling networks. Histological, electrochemical, and behavioral assays were performed in conditional knockout, small hairpin RNA–mediated knockdown, and AD-related mice models to evaluate the relevance of CDK5 to synaptic homeostasis and functions. Results Among candidates with known implications in synaptic modulations, BAG3 ranked the highest. CDK5-mediated phosphorylation on S297/S291 (mouse/human) destabilized BAG3. Loss of BAG3 unleashed the selective protein degradative function of the HSP70 machinery. In neurons, this resulted in enhanced degradation of a number of glutamatergic synaptic proteins. Conditional neuronal knockout of Bag3 in vivo led to impairment of learning and memory functions. In human AD and related mouse models, aberrant CDK5-mediated loss of BAG3 yielded similar effects on synaptic homeostasis. Detrimental effects of BAG3 loss on learning and memory functions were confirmed in these mice, and such effects were reversed by ectopic BAG3 reexpression. Conclusions Our results highlight that the neuronal CDK5-BAG3-HSP70 signaling axis plays a critical role in modulating synaptic homeostasis. Dysregulation of the signaling pathway directly contributes to synaptic dysfunction and AD pathogenesis.