Nanoparticle-Mediated Delivery of Anti-PU.1 siRNA via Localized Intracisternal Administration Reduces Neuroinflammation.

Neuroinflammation is a hallmark of neurodegenerative disorders including Alzheimer's disease (AD). Microglia, the brain's immune cells, express many of the AD-risk loci identified in genome wide association studies and present a promising target for anti-inflammatory RNA therapeutics but are difficult to transfect with current methods. Here, several lipid nanoparticle (LNP) formulations are examined, and a lead candidate that supports efficient RNA delivery in cultures of human stem cell-derived microglia-like cells (iMGLs) and animal models of neuroinflammation is identified. The lead microglia LNP (MG-LNP) formulation shows minimal toxicity and improves delivery efficiency to inflammatory iMGLs, suggesting a preference for delivery into activated microglia. Intraperitoneal injection of the MG-LNP formulation generates widespread expression of the delivered reporter construct in all organs, whereas local intracisternal injection directly into the cerebrospinal fluid leads to preferential expression in the brain. It is shown that LNP-mediated delivery of siRNA targeting the PU.1 transcription factor, a known AD-risk locus, successfully reduces PU.1 levels in iMGLs and reduces neuroinflammation in mice injected with LPS and in CK-p25 mice that mimic the chronic neuroinflammation seen in AD patients. The LNP formulation represents an effective RNA delivery vehicle when applied intrathecally and can be broadly utilized to test potential neuroinflammation-directed gene therapies.

iPSC-derived microglia carrying the TREM2 R47H/+ mutation are proinflammatory and promote synapse loss.

Genetic findings have highlighted key roles for microglia in the pathology of neurodegenerative conditions such as Alzheimer's disease (AD). A number of mutations in the microglial protein triggering receptor expressed on myeloid cells 2 (TREM2) have been associated with increased risk for developing AD, most notably the R47H/+ substitution. We employed gene editing and stem cell models to gain insight into the effects of the TREM2 R47H/+ mutation on human-induced pluripotent stem cell-derived microglia. We found transcriptional changes affecting numerous cellular processes, with R47H/+ cells exhibiting a proinflammatory gene expression signature. TREM2 R47H/+ also caused impairments in microglial movement and the uptake of multiple substrates, as well as rendering microglia hyperresponsive to inflammatory stimuli. We developed an in vitro laser-induced injury model in neuron-microglia cocultures, finding an impaired injury response by TREM2 R47H/+ microglia. Furthermore, mouse brains transplanted with TREM2 R47H/+ microglia exhibited reduced synaptic density, with upregulation of multiple complement cascade components in TREM2 R47H/+ microglia suggesting inappropriate synaptic pruning as one potential mechanism. These findings identify a number of potentially detrimental effects of the TREM2 R47H/+ mutation on microglial gene expression and function likely to underlie its association with AD.

A novel molecular class that recruits HDAC/MECP2 complexes to PU.1 motifs reduces neuroinflammation.

Pervasive neuroinflammation occurs in many neurodegenerative diseases, including Alzheimer's disease (AD). SPI1/PU.1 is a transcription factor located at a genome-wide significant AD-risk locus and its reduced expression is associated with delayed onset of AD. We analyzed single-cell transcriptomic datasets from microglia of human AD patients and found an enrichment of PU.1-binding motifs in the differentially expressed genes. In hippocampal tissues from transgenic mice with neurodegeneration, we found vastly increased genomic PU.1 binding. We then screened for PU.1 inhibitors using a PU.1 reporter cell line and discovered A11, a molecule with anti-inflammatory efficacy and nanomolar potency. A11 regulated genes putatively by recruiting a repressive complex containing MECP2, HDAC1, SIN3A, and DNMT3A to PU.1 motifs, thus representing a novel mechanism and class of molecules. In mouse models of AD, A11 ameliorated neuroinflammation, loss of neuronal integrity, AD pathology, and improved cognitive performance. This study uncovers a novel class of anti-inflammatory molecules with therapeutic potential for neurodegenerative disorders.

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.

Phosphoproteomics identifies microglial Siglec-F inflammatory response during neurodegeneration.

Alzheimer's disease (AD) is characterized by the appearance of amyloid-ß plaques, neurofibrillary tangles, and inflammation in brain regions involved in memory. Using mass spectrometry, we have quantified the phosphoproteome of the CK-p25, 5XFAD, and Tau P301S mouse models of neurodegeneration. We identified a shared response involving Siglec-F which was upregulated on a subset of reactive microglia. The human paralog Siglec-8 was also upregulated on microglia in AD. Siglec-F and Siglec-8 were upregulated following microglial activation with interferon gamma (IFNγ) in BV-2 cell line and human stem cell-derived microglia models. Siglec-F overexpression activates an endocytic and pyroptotic inflammatory response in BV-2 cells, dependent on its sialic acid substrates and immunoreceptor tyrosine-based inhibition motif (ITIM) phosphorylation sites. Related human Siglecs induced a similar response in BV-2 cells. Collectively, our results point to an important role for mouse Siglec-F and human Siglec-8 in regulating microglial activation during neurodegeneration.

HDAC1 modulates OGG1-initiated oxidative DNA damage repair in the aging brain and Alzheimer's disease.

DNA damage contributes to brain aging and neurodegenerative diseases. However, the factors stimulating DNA repair to stave off functional decline remain obscure. We show that HDAC1 modulates OGG1-initated 8-oxoguanine (8-oxoG) repair in the brain. HDAC1-deficient mice display age-associated DNA damage accumulation and cognitive impairment. HDAC1 stimulates OGG1, a DNA glycosylase known to remove 8-oxoG lesions that are associated with transcriptional repression. HDAC1 deficiency causes impaired OGG1 activity, 8-oxoG accumulation at the promoters of genes critical for brain function, and transcriptional repression. Moreover, we observe elevated 8-oxoG along with reduced HDAC1 activity and downregulation of a similar gene set in the 5XFAD mouse model of Alzheimer's disease. Notably, pharmacological activation of HDAC1 alleviates the deleterious effects of 8-oxoG in aged wild-type and 5XFAD mice. Our work uncovers important roles for HDAC1 in 8-oxoG repair and highlights the therapeutic potential of HDAC1 activation to counter functional decline in brain aging and neurodegeneration.

Modeling Alzheimer's disease with iPSC-derived brain cells.

Alzheimer's disease is a devastating neurodegenerative disorder with no cure. Countless promising therapeutics have shown efficacy in rodent Alzheimer's disease models yet failed to benefit human patients. While hope remains that earlier intervention with existing therapeutics will improve outcomes, it is becoming increasingly clear that new approaches to understand and combat the pathophysiology of Alzheimer's disease are needed. Human induced pluripotent stem cell (iPSC) technologies have changed the face of preclinical research and iPSC-derived cell types are being utilized to study an array of human conditions, including neurodegenerative disease. All major brain cell types can now be differentiated from iPSCs, while increasingly complex co-culture systems are being developed to facilitate neuroscience research. Many cellular functions perturbed in Alzheimer's disease can be recapitulated using iPSC-derived cells in vitro, and co-culture platforms are beginning to yield insights into the complex interactions that occur between brain cell types during neurodegeneration. Further, iPSC-based systems and genome editing tools will be critical in understanding the roles of the numerous new genes and mutations found to modify Alzheimer's disease risk in the past decade. While still in their relative infancy, these developing iPSC-based technologies hold considerable promise to push forward efforts to combat Alzheimer's disease and other neurodegenerative disorders.

Examining the Role of HDACs in DNA Double-Strand Break Repair in Neurons.

Histone deacetylases (HDACs) modulate chromatin structure by removing acetyl groups from histones. Upon DNA double-strand breaks (DSBs), deacetylation of H3K56 and H4K16 by HDACs occurs immediately at break sites, and is crucial for DSB repair. Here we describe two assays that examine defective DSB repair caused by HDAC inhibition in primary cortical neurons: single-cell gel electrophoresis to assay DNA integrity (the comet assay) and western blot analysis for γH2AX, a phosphorylated histone variant associated with DSBs.

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.

A Developmental Switch in Microglial HDAC Function.

The epigenetic mechanisms controlling microglia functions are largely unknown. In this issue of Immunity, Datta et al. (2018) uncover surprising and distinct effects of deleting the epigenetic regulators HDAC1 and HDAC2 during microglial development versus during the course of neurodegeneration.

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.

Acute Fasting Regulates Retrograde Synaptic Enhancement through a 4E-BP-Dependent Mechanism.

While beneficial effects of fasting on organismal function and health are well appreciated, we know little about the molecular details of how fasting influences synaptic function and plasticity. Our genetic and electrophysiological experiments demonstrate that acute fasting blocks retrograde synaptic enhancement that is normally triggered as a result of reduction in postsynaptic receptor function at the Drosophila larval neuromuscular junction (NMJ). This negative regulation critically depends on transcriptional enhancement of eukaryotic initiation factor 4E binding protein (4E-BP) under the control of the transcription factor Forkhead box O (Foxo). Furthermore, our findings indicate that postsynaptic 4E-BP exerts a constitutive negative input, which is counteracted by a positive regulatory input from the Target of Rapamycin (TOR). This combinatorial retrograde signaling plays a key role in regulating synaptic strength. Our results provide a mechanistic insight into how cellular stress and nutritional scarcity could acutely influence synaptic homeostasis and functional stability in neural circuits.

The road to restoring neural circuits for the treatment of Alzheimer's disease.

Alzheimer's disease is a progressive loss of memory and cognition, for which there is no cure. Although genetic studies initially suggested a primary role for amyloid-in Alzheimer's disease, treatment strategies targeted at reducing amyloid-have failed to reverse cognitive symptoms. These clinical findings suggest that cognitive decline is the result of a complex pathophysiology and that targeting amyloid-alone may not be sufficient to treat Alzheimer's disease. Instead, a broad outlook on neural-circuit-damaging processes may yield insights into new therapeutic strategies for curing memory loss in the disease.

LRRK2 regulates retrograde synaptic compensation at the Drosophila neuromuscular junction.

Parkinson's disease gene leucine-rich repeat kinase 2 (LRRK2) has been implicated in a number of processes including the regulation of mitochondrial function, autophagy and endocytic dynamics; nevertheless, we know little about its potential role in the regulation of synaptic plasticity. Here we demonstrate that postsynaptic knockdown of the fly homologue of LRRK2 thwarts retrograde, homeostatic synaptic compensation at the larval neuromuscular junction. Conversely, postsynaptic overexpression of either the fly or human LRRK2 transgene induces a retrograde enhancement of presynaptic neurotransmitter release by increasing the size of the release ready pool of vesicles. We show that LRRK2 promotes cap-dependent translation and identify Furin 1 as its translational target, which is required for the synaptic function of LRRK2. As the regulation of synaptic homeostasis plays a fundamental role in ensuring normal and stable synaptic function, our findings suggest that aberrant function of LRRK2 may lead to destabilization of neural circuits.

JAKMIP1: Translating the Message for Social Behavior.

In this issue of Neuron, Berg et al. (2015) uncover multifaceted roles for janus kinase and microtubule-interacting protein 1 (JAKMIP1) in regulating neuronal mRNA translation and establish JAKMIP1 knockout mice as an important model to study autism spectrum disorder-associated phenotypes.

Histone deacetylases in memory and cognition.

Over the past 30 years, lysine acetylation of histone and nonhistone proteins has become established as a key modulator of gene expression regulating numerous aspects of cell biology. Neuronal growth and plasticity are no exception; roles for lysine acetylation and deacetylation in brain function and dysfunction continue to be uncovered. Transcriptional programs coupling synaptic activity to changes in gene expression are critical to the plasticity mechanisms underlying higher brain functions. These transcriptional programs can be modulated by changes in histone acetylation, and in many cases, transcription factors and histone-modifying enzymes are recruited together to plasticity-associated genes. Lysine acetylation, catalyzed by lysine acetyltransferases (KATs), generally promotes cognitive performance, whereas the opposing process, catalyzed by histone lysine deacetylases (HDACs), appears to negatively regulate cognition in multiple brain regions. Consistently, mutation or deregulation of different KATs or HDACs contributes to neurological dysfunction and neurodegeneration. HDAC inhibitors have shown promise as a treatment to combat the cognitive decline associated with aging and neurodegenerative disease, as well as to ameliorate the symptoms of depression and posttraumatic stress disorder, among others. In this review, we discuss the evidence for the roles of HDACs in cognitive function as well as in neurological disorders and disease. In particular, we focus on HDAC2, which plays a central role in coupling lysine acetylation to synaptic plasticity and mediates many of the effects of HDAC inhibition in cognition and disease.