A Cdk5-derived peptide inhibits Cdk5/p25 activity and improves neurodegenerative phenotypes.

Aberrant activity of cyclin-dependent kinase (Cdk5) has been implicated in various neurodegenerative diseases. This deleterious effect is mediated by pathological cleavage of the Cdk5 activator p35 into the truncated product p25, leading to prolonged Cdk5 activation and altered substrate specificity. Elevated p25 levels have been reported in humans and rodents with neurodegeneration, and the benefit of genetically blocking p25 production has been demonstrated previously in rodent and human neurodegenerative models. Here, we report a 12-amino-acid-long peptide fragment derived from Cdk5 (Cdk5i) that is considerably smaller than existing peptide inhibitors of Cdk5 (P5 and CIP) but shows high binding affinity toward the Cdk5/p25 complex, disrupts the interaction of Cdk5 with p25, and lowers Cdk5/p25 kinase activity. When tagged with a fluorophore (FITC) and the cell-penetrating transactivator of transcription (TAT) sequence, the Cdk5i-FT peptide exhibits cell- and brain-penetrant properties and confers protection against neurodegenerative phenotypes associated with Cdk5 hyperactivity in cell and mouse models of neurodegeneration, highlighting Cdk5i's therapeutic potential.

Calcineurin dephosphorylates topoisomerase IIß and regulates the formation of neuronal-activity-induced DNA breaks.

Neuronal activity induces topoisomerase IIß (Top2B) to generate DNA double-strand breaks (DSBs) within the promoters of neuronal early response genes (ERGs) and facilitate their transcription, and yet, the mechanisms that control Top2B-mediated DSB formation are unknown. Here, we report that stimulus-dependent calcium influx through NMDA receptors activates the phosphatase calcineurin to dephosphorylate Top2B at residues S1509 and S1511, which stimulates its DNA cleavage activity and induces it to form DSBs. Exposing mice to a fear conditioning paradigm also triggers Top2B dephosphorylation at S1509 and S1511 in the hippocampus, indicating that calcineurin also regulates Top2B-mediated DSB formation following physiological neuronal activity. Furthermore, calcineurin-Top2B interactions following neuronal activity and sites that incur activity-induced DSBs are preferentially localized at the nuclear periphery in neurons. Together, these results reveal how radial gene positioning and the compartmentalization of activity-dependent signaling govern the position and timing of activity-induced DSBs and regulate gene expression patterns in neurons.

Multi-sensory Gamma Stimulation Ameliorates Alzheimer's-Associated Pathology and Improves Cognition.

We previously reported that inducing gamma oscillations with a non-invasive light flicker (gamma entrainment using sensory stimulus or GENUS) impacted pathology in the visual cortex of Alzheimer's disease mouse models. Here, we designed auditory tone stimulation that drove gamma frequency neural activity in auditory cortex (AC) and hippocampal CA1. Seven days of auditory GENUS improved spatial and recognition memory and reduced amyloid in AC and hippocampus of 5XFAD mice. Changes in activation responses were evident in microglia, astrocytes, and vasculature. Auditory GENUS also reduced phosphorylated tau in the P301S tauopathy model. Furthermore, combined auditory and visual GENUS, but not either alone, produced microglial-clustering responses, and decreased amyloid in medial prefrontal cortex. Whole brain analysis using SHIELD revealed widespread reduction of amyloid plaques throughout neocortex after multi-sensory GENUS. Thus, GENUS can be achieved through multiple sensory modalities with wide-ranging effects across multiple brain areas to improve cognitive function.

Author Correction: Gamma frequency entrainment attenuates amyloid load and modifies microglia.

Change history: In this Article, Extended Data Fig. 8 and Extended Data Table 1 contained errors, which have been corrected online.

APOE4 Causes Widespread Molecular and Cellular Alterations Associated with Alzheimer's Disease Phenotypes in Human iPSC-Derived Brain Cell Types.

The apolipoprotein E4 (APOE4) variant is the single greatest genetic risk factor for sporadic Alzheimer's disease (sAD). However, the cell-type-specific functions of APOE4 in relation to AD pathology remain understudied. Here, we utilize CRISPR/Cas9 and induced pluripotent stem cells (iPSCs) to examine APOE4 effects on human brain cell types. Transcriptional profiling identified hundreds of differentially expressed genes in each cell type, with the most affected involving synaptic function (neurons), lipid metabolism (astrocytes), and immune response (microglia-like cells). APOE4 neurons exhibited increased synapse number and elevated Aß42 secretion relative to isogenic APOE3 cells while APOE4 astrocytes displayed impaired Aß uptake and cholesterol accumulation. Notably, APOE4 microglia-like cells exhibited altered morphologies, which correlated with reduced Aß phagocytosis. Consistently, converting APOE4 to APOE3 in brain cell types from sAD iPSCs was sufficient to attenuate multiple AD-related pathologies. Our study establishes a reference for human cell-type-specific changes associated with the APOE4 variant. VIDEO ABSTRACT.

Loss of Protein Arginine Methyltransferase 8 Alters Synapse Composition and Function, Resulting in Behavioral Defects.

Diverse molecular mechanisms regulate synaptic composition and function in the mammalian nervous system. The multifunctional protein arginine methyltransferase 8 (PRMT8) possesses both methyltransferase and phospholipase activities. Here we examine the role of this neuron-specific protein in hippocampal plasticity and cognitive function. PRMT8 protein localizes to synaptic sites, and conditional whole-brain Prmt8 deletion results in altered levels of multiple synaptic proteins in the hippocampus, using both male and female mice. Interestingly, these altered protein levels are due to post-transcriptional mechanisms as the corresponding mRNA levels are unaffected. Strikingly, electrophysiological recordings from hippocampal slices of mice lacking PRMT8 reveal multiple defects in excitatory synaptic function and plasticity. Furthermore, behavioral analyses show that PRMT8 conditional knock-out mice exhibit impaired hippocampal-dependent fear learning. Together, these findings establish PRMT8 as an important component of the molecular machinery required for hippocampal neuronal function.SIGNIFICANCE STATEMENT Numerous molecular processes are critically required for normal brain function. Here we use mice lacking protein arginine methyltransferase 8 (PRMT8) in the brain to examine how loss of this protein affects the structure and function of neurons in the hippocampus. We find that PRMT8 localizes to the sites of communication between neurons. Hippocampal neurons from mice lacking PRMT8 have no detectable structural differences compared with controls; however, multiple aspects of their function are altered. Consistently, we find that mice lacking PRMT8 also exhibit reduced hippocampus-dependent memory. Together, our findings establish important roles for PRMT8 in regulating neuron function and cognition in the mammalian brain.

Inhibition of p25/Cdk5 Attenuates Tauopathy in Mouse and iPSC Models of Frontotemporal Dementia.

Increased p25, a proteolytic fragment of the regulatory subunit p35, is known to induce aberrant activity of cyclin-dependent kinase 5 (Cdk5), which is associated with neurodegenerative disorders, including Alzheimer's disease. Previously, we showed that replacing endogenous p35 with the noncleavable mutant p35 (Δp35) attenuated amyloidosis and improved cognitive function in a familial Alzheimer's disease mouse model. Here, to address the role of p25/Cdk5 in tauopathy, we generated double-transgenic mice by crossing mice overexpressing mutant human tau (P301S) with Δp35KI mice. We observed significant reduction of phosphorylated tau and its seeding activity in the brain of double transgenic mice compared with the P301S mice. Furthermore, synaptic loss and impaired LTP at hippocampal CA3 region of P301S mice were attenuated by blocking p25 generation. To further validate the role of p25/Cdk5 in tauopathy, we used frontotemporal dementia patient-derived induced pluripotent stem cells (iPSCs) carrying the Tau P301L mutation and generated P301L:Δp35KI isogenic iPSC lines using CRISPR/Cas9 genome editing. We created cerebral organoids from the isogenic iPSCs and found that blockade of p25 generation reduced levels of phosphorylated tau and increased expression of synaptophysin. Together, these data demonstrate a crucial role for p25/Cdk5 in mediating tau-associated pathology and suggest that inhibition of this kinase can remedy neurodegenerative processes in the presence of pathogenic tau mutation.SIGNIFICANCE STATEMENT Accumulation of p25 results in aberrant Cdk5 activation and induction of numerous pathological phenotypes, such as neuroinflammation, synaptic loss, Aß accumulation, and tau hyperphosphorylation. However, it was not clear whether p25/Cdk5 activity is necessary for the progression of these pathological changes. We recently developed the Δp35KI transgenic mouse that is deficient in p25 generation and Cdk5 hyperactivation. In this study, we used this mouse model to elucidate the role of p25/Cdk5 in FTD mutant tau-mediated pathology. We also used a frontotemporal dementia patient-derived induced pluripotent stem cell carrying the Tau P301L mutation and generated isogenic lines in which p35 is replaced with noncleavable mutant Δp35. Our data suggest that p25/Cdk5 plays an important role in tauopathy in both mouse and human model systems.

The Transcription Factor Sp3 Cooperates with HDAC2 to Regulate Synaptic Function and Plasticity in Neurons.

The histone deacetylase HDAC2, which negatively regulates synaptic gene expression and neuronal plasticity, is upregulated in Alzheimer's disease (AD) patients and mouse models. Therapeutics targeting HDAC2 hold promise for ameliorating AD-related cognitive impairment; however, attempts to generate HDAC2-specific inhibitors have failed. Here, we take an integrative genomics approach to identify proteins that mediate HDAC2 recruitment to synaptic plasticity genes. Functional screening revealed that knockdown of the transcription factor Sp3 phenocopied HDAC2 knockdown and that Sp3 facilitated recruitment of HDAC2 to synaptic genes. Importantly, like HDAC2, Sp3 expression was elevated in AD patients and mouse models, where Sp3 knockdown ameliorated synaptic dysfunction. Furthermore, exogenous expression of an HDAC2 fragment containing the Sp3-binding domain restored synaptic plasticity and memory in a mouse model with severe neurodegeneration. Our findings indicate that targeting the HDAC2-Sp3 complex could enhance cognitive function without affecting HDAC2 function in other processes.

Gamma frequency entrainment attenuates amyloid load and modifies microglia.

Changes in gamma oscillations (20-50 Hz) have been observed in several neurological disorders. However, the relationship between gamma oscillations and cellular pathologies is unclear. Here we show reduced, behaviourally driven gamma oscillations before the onset of plaque formation or cognitive decline in a mouse model of Alzheimer's disease. Optogenetically driving fast-spiking parvalbumin-positive (FS-PV)-interneurons at gamma (40 Hz), but not other frequencies, reduces levels of amyloid-ß (Aß)1-40 and Aß 1-42 isoforms. Gene expression profiling revealed induction of genes associated with morphological transformation of microglia, and histological analysis confirmed increased microglia co-localization with Aß. Subsequently, we designed a non-invasive 40 Hz light-flickering regime that reduced Aß1-40 and Aß1-42 levels in the visual cortex of pre-depositing mice and mitigated plaque load in aged, depositing mice. Our findings uncover a previously unappreciated function of gamma rhythms in recruiting both neuronal and glial responses to attenuate Alzheimer's-disease-associated pathology.