The Amyloid Precursor Protein Modulates the Position and Length of the Axon Initial Segment.

The amyloid precursor protein (APP) is linked to the genetics and pathogenesis of Alzheimer's disease (AD). It is the parent protein of the ß-amyloid (Aß) peptide, the main constituent of the amyloid plaques found in an AD brain. The pathways from APP to Aß 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 in App gene 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 of App block the glutamate effects. The actions of APP on the AIS are cell-autonomous; exogenous Aß, 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 ßIV-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 vivo as in 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 STATEMENT While 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 vivo and in vitro, modest amounts of excess APP alter the properties of the axon initial segment. The ß-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.

Apolipoprotein E ε4 disrupts oligodendrocyte differentiation by interfering with astrocyte-derived lipid transport.

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 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) knock-in 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+ ) and an abnormal accumulation of OL precursors (Nkx 2.2+ ), suggesting a disruption of OL differentiation. Gene ontology analysis of an existing RNA-seq dataset confirms a robust transcriptional response to the altered chemistry of the hAPOE4 mouse brain. In culture, the uptake of astrocyte-derived APOE during Lovastatin-mediated depletion of cholesterol synthesis is sufficient to sustain OL differentiation. While endogenous hAPOE protein isoforms have no effects on OL development, exogenous hAPOE4 abolishes the ability of very low-density lipoprotein to restore myelination in Apoe-deficient, cholesterol-depleted OL. Our data suggest that APOE4 impairs myelination in the aging brain by interrupting the delivery of astrocyte-derived lipids to the oligodendrocytes. We propose that high myelin turnover and OL exhaustion found in APOE4 carriers is a likely explanation for the APOE-dependent myelin phenotypes of the AD brain.

DNA Repair Inhibition Leads to Active Export of Repetitive Sequences to the Cytoplasm Triggering an Inflammatory Response.

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 and Atm-/- 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 STATEMENT Sterile 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.

Asymmetric left-right hippocampal glutamatergic modulation of cognitive control in ApoE-isoform subjects is unrelated to neuroinflammation.

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.

Small-World Networks and Their Relationship With Hippocampal Glutamine/Glutamate Concentration in Healthy Adults With Varying Genetic Risk for Alzheimer's Disease.

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 (Ci ) and nodal characteristic path length (Li )) 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 < 0.05) in Ci between subgroups were found in hubs of left inferior frontal, bilateral inferior temporal, and bilateral precentral gyri, right parahippocampus, and bilateral precuneus. In addition, there was a significant correlation between Glx in the left hippocampus and Ci in inferior frontal gyrus (r = -0.537, P = 0.024), right inferior temporal (r = -0.478, P = 0.043), right parahippocampus (r = -0.629, P = 0.016), left precentral (r = -0.581, P = 0.022), right precentral (r = -0.651, P = 0.003), left precuneus (r = -0.545, P = 0.024), and right precuneus (r = -0.567, P = 0.022); and Li in left precuneus (r = 0.575, P = 0.032) and right precuneus (r = 0.586, P = 0.032) in the high-risk group, but not in the low-risk group. DATA CONCLUSION: Our results suggested that healthy ApoE4 carriers exhibit poorer local interconnectivity. Moreover, the close relationship between glutamate and small-world network properties in ApoE4 carriers might reflect a compensatory response to the impaired network efficiency. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 3.

Selective loss of 5hmC promotes neurodegeneration in the mouse model of Alzheimer's disease.

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 Aß. 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 Aß 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.

ATM and ATR play complementary roles in the behavior of excitatory and inhibitory vesicle populations.

ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related) are large PI3 kinases whose human mutations result in complex syndromes that include a compromised DNA damage response (DDR) and prominent nervous system phenotypes. Both proteins are nuclear-localized in keeping with their DDR functions, yet both are also found in cytoplasm, including on neuronal synaptic vesicles. In ATM- or ATR-deficient neurons, spontaneous vesicle release is reduced, but a drop in ATM or ATR level also slows FM4-64 dye uptake. In keeping with this, both proteins bind to AP-2 complex components as well as to clathrin, suggesting roles in endocytosis and vesicle recycling. The two proteins play complementary roles in the DDR; ATM is engaged in the repair of double-strand breaks, while ATR deals mainly with single-strand damage. Unexpectedly, this complementarity extends to these proteins' synaptic function as well. Superresolution microscopy and coimmunoprecipitation reveal that ATM associates exclusively with excitatory (VGLUT1+) vesicles, while ATR associates only with inhibitory (VGAT+) vesicles. The levels of ATM and ATR respond to each other; when ATM is deficient, ATR levels rise, and vice versa. Finally, blocking NMDA, but not GABA, receptors causes ATM levels to rise while ATR levels respond to GABA, but not NMDA, receptor blockade. Taken together, our data suggest that ATM and ATR are part of the cellular "infrastructure" that maintains the excitatory/inhibitory balance of the nervous system. This idea has important implications for the human diseases resulting from their genetic deficiency.

Accumulation of Cytoplasmic DNA Due to ATM Deficiency Activates the Microglial Viral Response System with Neurotoxic Consequences.

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-1ß. 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.

Marine bacterial extracts as a new rich source of drugs against Alzheimer's disease.

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.

Genomic integrity and the ageing brain.

DNA damage is correlated with and may drive the ageing process. Neurons in the brain are postmitotic and are excluded from many forms of DNA repair; therefore, neurons are vulnerable to various neurodegenerative diseases. The challenges facing the field are to understand how and when neuronal DNA damage accumulates, how this loss of genomic integrity might serve as a 'time keeper' of nerve cell ageing and why this process manifests itself as different diseases in different individuals.

Testing the Neuroprotective Properties of PCSO-524® Using a Neuronal Cell Cycle Suppression Assay.

Cell cycle reentry is a unified mechanism shared by several neurodegenerative diseases, including Alzheimer's disease (AD) and Ataxia Telangiectasia (A-T). This phenotype is often related to neuroinflammation in the central nervous system. To mimic brain inflammation in vitro, we adopted the previously established method of using conditioned medium collected from activated THP-1 cells and applied it to both differentiated HT22 cells and primary neurons. Unscheduled cell cycle events were observed in both systems, indicating the potential of this approach as an in vitro model of neurodegenerative disease. We used this assay to measure the neuroprotective effects of New Zealand green-lipped mussel extract, PCSO-524®, to protect post-mitotic cells from cell cycle reentry. We found that, both in vitro and in an animal model, PCSO-524® displayed promising neuroprotective effects, and thus has potential to postpone or prevent the onset of neurodegenerative disease.

Ibuprofen prevents progression of ataxia telangiectasia symptoms in ATM-deficient mice.

BACKGROUND: Inflammation plays a critical role in accelerating the progression of neurodegenerative diseases, such as Alzheimer's disease (AD) and ataxia telangiectasia (A-T). In A-T mouse models, LPS-induced neuroinflammation advances the degenerative changes found in cerebellar Purkinje neurons both in vivo and in vitro. In the current study, we ask whether ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), can have the opposite effect and delay the symptoms of the disease. METHODS: We tested the beneficial effects of ibuprofen in both in vitro and in vivo models. Conditioned medium from LPS stimulated primary microglia (LM) applied to cultures of dissociated cortical neurons leads to numerous degenerative changes. Pretreatment of the neurons with ibuprofen, however, blocked this damage. Systemic injection of LPS into either adult wild-type or adult Atm-/- mice produced an immune challenge that triggered profound behavioral, biochemical, and histological effects. We used a 2-week ibuprofen pretreatment regimen to investigate whether these LPS effects could be blocked. We also treated young presymptomatic Atm-/- mice to determine if ibuprofen could delay the appearance of symptoms. RESULTS: Adding ibuprofen directly to neuronal cultures significantly reduced LM-induced degeneration. Curiously, adding ibuprofen to the microglia cultures before the LPS challenge had little effect, thus implying a direct effect of the NSAID on the neuronal cultures. In vivo administration of ibuprofen to Atm-/- animals before a systemic LPS immune challenge suppressed cytological damage. The ibuprofen effects were widespread as microglial activation, p38 phosphorylation, DNA damage, and neuronal cell cycle reentry were all reduced. Unfortunately, ibuprofen only slightly improved the LPS-induced behavioral deficits. Yet, while the behavioral symptoms could not be reversed once they were established in adult Atm-/- animals, administration of ibuprofen to young mutant pups prevented their symptoms from appearing. CONCLUSION: Inflammatory processes impact the normal progression of A-T implying that modulation of the immune system can have therapeutic benefit for both the behavioral and cellular symptoms of this neurodegenerative disease.

DNA damage-associated oligodendrocyte degeneration precedes amyloid pathology and contributes to Alzheimer's disease and dementia.

INTRODUCTION: In looking for novel non-amyloid-based etiologies for Alzheimer's disease, we explore the hypothesis that age-related myelin loss is an attractive explanation for age-associated cognitive decline and dementia. METHODS: We performed a meta-analysis of data in the National Alzheimer's Coordinating Center database accompanied by quantitative histopathology of myelin and oligodendrocytes (OLs) in frontal cortices of 24 clinically characterized individuals. Pathological findings were further validated in an Alzheimer's disease mouse model and in culture. RESULTS: Myelin lesions increased with cognitive impairment in an amyloid-independent fashion with signs of degeneration appearing before neuronal loss. Myelinating OLs in the gray matter showed greater vulnerability than those in white matter, and the degenerative changes correlated with evidence of DNA damage. Similar results were found in myelinating OL cultures where DNA damage caused aberrant OL cell cycle re-entry and death. DISCUSSION: We present the first comprehensive analysis of the cell biology of early myelin loss in sporadic Alzheimer's disease.

Family-wide Structural Characterization and Genomic Comparisons Decode the Diversity-oriented Biosynthesis of Thalassospiramides by Marine Proteobacteria.

The thalassospiramide lipopeptides have great potential for therapeutic applications; however, their structural and functional diversity and biosynthesis are poorly understood. Here, by cultivating 130 Rhodospirillaceae strains sampled from oceans worldwide, we discovered 21 new thalassospiramide analogues and demonstrated their neuroprotective effects. To investigate the diversity of biosynthetic gene cluster (BGC) architectures, we sequenced the draft genomes of 28 Rhodospirillaceae strains. Our family-wide genomic analysis revealed three types of dysfunctional BGCs and four functional BGCs whose architectures correspond to four production patterns. This correlation allowed us to reassess the "diversity-oriented biosynthesis" proposed for the microbial production of thalassospiramides, which involves iteration of several key modules. Preliminary evolutionary investigation suggested that the functional BGCs could have arisen through module/domain loss, whereas the dysfunctional BGCs arose through horizontal gene transfer. Further comparative genomics indicated that thalassospiramide production is likely to be attendant on particular genes/pathways for amino acid metabolism, signaling transduction, and compound efflux. Our findings provide a systematic understanding of thalassospiramide production and new insights into the underlying mechanism.

Re-imagining Alzheimer's disease - the diminishing importance of amyloid and a glimpse of what lies ahead.

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.

CDK5 activator protein p25 preferentially binds and activates GSK3ß.

Glycogen synthase kinase 3ß (GSK3ß) and cyclin-dependent kinase 5 (CDK5) are tau kinases and have been proposed to contribute to the pathogenesis of Alzheimer's disease. The 3D structures of these kinases are remarkably similar, which led us to hypothesize that both might be capable of binding cyclin proteins--the activating cofactors of all CDKs. CDK5 is normally activated by the cyclin-like proteins p35 and p39. By contrast, we show that GSK3ß does not bind to p35 but unexpectedly binds to p25, the calpain cleavage product of p35. Indeed, overexpressed GSK3ß outcompetes CDK5 for p25, whereas CDK5 is the preferred p35 partner. FRET analysis reveals nanometer apposition of GSK3ß:p25 in cell soma as well as in synaptic regions. Interaction with p25 also alters GSK3ß substrate specificity. The GSK3ß:p25 interaction leads to enhanced phosphorylation of tau, but decreased phosphorylation of ß-catenin. A partial explanation for this situation comes from in silico modeling, which predicts that the docking site for p25 on GSK3ß is the AXIN-binding domain; because of this, p25 inhibits the formation of the GSK3ß/AXIN/APC destruction complex, thus preventing GSK3ß from binding to and phosphorylating ß-catenin. Coexpression of GSK3ß and p25 in cultured neurons results in a neurodegeneration phenotype that exceeds that observed with CDK5 and p25. When p25 is transfected alone, the resulting neuronal damage is blocked more effectively with a specific siRNA against Gsk3ß than with one against Cdk5. We propose that the effects of p25, although normally attributed to activate CDK5, may be mediated in part by elevated GSK3ß activity.

The roles of Cdk5-mediated subcellular localization of FOXO1 in neuronal death.

Deficiency of cyclin-dependent kinase 5 (Cdk5) has been linked to the death of postmitotic cortical neurons during brain development. We now report that, in mouse cortical neurons, Cdk5 is capable of phosphorylating the transcription factor FOXO1 at Ser249 in vitro and in vivo. Cellular stresses resulting from extracellular stimulation by H2O2 or ß-amyloid promote hyperactivation of Cdk5, FOXO1 nuclear export and inhibition of its downstream transcriptional activity. In contrast, a loss of Cdk5 leads to FOXO1 translocation into the nucleus: a shift due to decreased AKT activity but independent of S249 phosphorylation. Nuclear FOXO1 upregulates transcription of the proapoptotic gene, BIM, leading to neuronal death, which can be rescued when endogenous FOXO1 was replaced by the cytoplasmically localized form of FOXO1, FOXO1-S249D. Cytoplasmic, but not nuclear, Cdk5 attenuates neuronal death by inhibiting FOXO1 transcriptional activity and BIM expression. Together, our findings suggest that Cdk5 plays a novel and unexpected role in the degeneration of postmitotic neurons through modulation of the cellular location of FOXO1, which constitutes an alternative pathway through which Cdk5 deficiency leads to neuronal death.

ATM protein is located on presynaptic vesicles and its deficit leads to failures in synaptic plasticity.

Ataxia telangiectasia is a multisystemic disorder that includes a devastating neurodegeneration phenotype. The ATM (ataxia-telangiectasia mutated) protein is well-known for its role in the DNA damage response, yet ATM is also found in association with cytoplasmic vesicular structures: endosomes and lysosomes, as well as neuronal synaptic vesicles. In keeping with this latter association, electrical stimulation of the Schaffer collateral pathway in hippocampal slices from ATM-deficient mice does not elicit normal long-term potentiation (LTP). The current study was undertaken to assess the nature of this deficit. Theta burst-induced LTP was reduced in Atm(-/-) animals, with the reduction most pronounced at burst stimuli that included 6 or greater trains. To assess whether the deficit was associated with a pre- or postsynaptic failure, we analyzed paired-pulse facilitation and found that it too was significantly reduced in Atm(-/-) mice. This indicates a deficit in presynaptic function. As further evidence that these synaptic effects of ATM deficiency were presynaptic, we used stochastic optical reconstruction microscopy. Three-dimensional reconstruction revealed that ATM is significantly more closely associated with Piccolo (a presynaptic marker) than with Homer1 (a postsynaptic marker). These results underline how, in addition to its nuclear functions, ATM plays an important functional role in the neuronal synapse where it participates in the regulation of presynaptic vesicle physiology.

Alteration in 5-hydroxymethylcytosine-mediated epigenetic regulation leads to Purkinje cell vulnerability in ATM deficiency.

A long-standing mystery surrounding ataxia-telangiectasia is why it is mainly cerebellar neurons, Purkinje cells in particular, that appear vulnerable to ATM deficiency. Here we present data showing that 5-hydroxymethylcytosine (5hmC), a newly recognized epigenetic marker found at high levels in neurons, is substantially reduced in human ataxia-telangiectasia and Atm(-/-) mouse cerebellar Purkinje cells. We further show that TET1, an enzyme that converts 5-methylcytosine (5mC) to 5hmC, responds to DNA damage and manipulation of TET1 activity directly affects the DNA damage signalling and ATM-deficient neuronal cell cycle re-entry and death. Quantitative genome-wide analysis of 5hmC-containing sequences shows that in ATM deficiency there is a cerebellum- and Purkinje cell-specific shift in 5hmC enrichment in both regulatory elements and repeated sequences. Finally, we verify that TET1-mediated 5hmC production is linked to the degenerative process of Purkinje cells and behavioural deficits in Atm(-/-) mice. Taken together, the selective loss of 5hmC plays a critical role in driving Purkinje cell vulnerability in ATM deficiency.