Small-molecule Klotho enhancers as novel treatment of neurodegeneration

The majority of neurodegenerative diseases have an important age component, and thus, understanding the molecular changes that occur during normal aging of the brain is of utmost relevance. In search for the basis of the age-related cognitive decline found in humans, monkeys and rodents, we study the rhesus monkey. Surprisingly, there is no loss of neurons in aged monkey brains. However, we reported white matter and myelin abnormalities in aged monkeys, similar to those observed in Alzheimer’s disease and multiple sclerosis patients. In a microarray analysis comparing young and old monkey white matter, we discovered that Klotho is downregulated in the aged brain. We then asked whether there is a connection between the age-related cognitive decline, myelin abnormalities and Klotho downregulation. If such a connection is found, compounds that upregulate Klotho expression could become of therapeutic interest for the treatment of multiple sclerosis, and perhaps even Alzheimer’s disease

Identification of novel small molecules that elevate Klotho expression

The absence of Klotho (KL) from mice causes the development of disorders associated with human aging and decreased longevity, whereas increased expression prolongs lifespan. With age, KL protein levels decrease, and keeping levels consistent may promote healthier aging and be disease-modifying. Using the KL promoter to drive expression of luciferase, we conducted a high-throughput screen to identify compounds that activate KL transcription. Hits were identified as compounds that elevated luciferase expression at least 30%. Following validation for dose-dependent activation and lack of cytotoxicity, hit compounds were evaluated further in vitro by incubation with opossum kidney and Z310 rat choroid plexus cells, which express KL endogenously. All compounds elevated KL protein compared with control. To determine whether increased protein resulted in an in vitro functional change, we assayed FGF23 (fibroblast growth factor 23) signalling. Compounds G–I augmented ERK (extracellular-signal-regulated kinase) phosphorylation in FGFR (fibroblast growth factor receptor)-transfected cells, whereas co-transfection with KL siRNA (small interfering RNA) blocked the effect. These compounds will be useful tools to allow insight into the mechanisms of KL regulation. Further optimization will provide pharmacological tools for in vivo studies of KL

Lowering of amyloid beta peptide production with a small molecule inhibitor of amyloid-? precursor protein dimerization

The amyloid ? precursor protein (APP) is a single-pass transmembrane glycoprotein that is ubiquitously expressed in many cell types, including neurons. Amyloidogenic processing of APP by ?- and ?-secretases leads to the production of amyloid-? (A?) peptides that can oligomerize and aggregate into amyloid plaques, a characteristic hallmark of Alzheimer’s disease (AD) brains. Multiple reports suggest that dimerization of APP may play a role in A? production; however, it is not yet clear whether APP dimers increase or decrease A? and the mechanism is not fully understood. To better understand the relationship between APP dimerization and production of A?, a high throughput screen for small molecule modulators of APP dimerization was conducted using APP-Firefly luciferase enzyme complementation to detect APP dimerization. Selected modulators identified from a compound library of 77,440 compounds were tested for their effects on A? generation. Two molecules that inhibited APP dimerization produced a reduction in A? levels as measured by ELISA. The inhibitors did not change sAPP? or ?-CTF levels, but lowered sAPP? levels, suggesting that blocking the dimerization is preventing the cleavage by ?-secretase in the amyloidogenic processing of APP. To our knowledge, this is the first High Throughput Screen (HTS) effort to identify small molecule modulators of APP dimerization. Inhibition of APP dimerization has previously been suggested as a therapeutic target in AD. The findings reported here further support that modulation of APP dimerization may be a viable means of reducing the production of A?

PLXNA4 is associated with Alzheimer disease and modulates tau phosphorylation

OBJECTIVE: Much of the genetic basis for Alzheimer disease (AD) is unexplained. We sought to identify novel AD loci using a unique family-based approach that can detect robust associations with infrequent variants (minor allele frequency < 0.10). METHODS: We conducted a genome-wide association study in the Framingham Heart Study (discovery) and NIA-LOAD (National Institute on Aging-Late-Onset Alzheimer Disease) Study (replication) family-based cohorts using an approach that accounts for family structure and calculates a risk score for AD as the outcome. Links between the most promising gene candidate and AD pathogenesis were explored in silico as well as experimentally in cell-based models and in human brain. RESULTS: Genome-wide significant association was identified with a PLXNA4 single nucleotide polymorphism (rs277470) located in a region encoding the semaphorin-3A (SEMA3A) binding domain (meta-analysis p value [meta-P] = 4.1 × 10(-8) ). A test for association with the entire region was also significant (meta-P = 3.2 × 10(-4) ). Transfection of SH-SY5Y cells or primary rat neurons with full-length PLXNA4 (TS1) increased tau phosphorylation with stimulated by SEMA3A. The opposite effect was observed when cells were transfected with shorter isoforms (TS2 and TS3). However, transfection of any isoform into HEK293 cells stably expressing amyloid β (Aβ) precursor protein (APP) did not result in differential effects on APP processing or Aβ production. Late stage AD cases (n = 9) compared to controls (n = 5) had 1.9-fold increased expression of TS1 in cortical brain tissue (p = 1.6 × 10(-4) ). Expression of TS1 was significantly correlated with the Clinical Dementia Rating score (ρ = 0.75, p = 2.2 × 10(-4) ), plaque density (ρ = 0.56, p = 0.01), and Braak stage (ρ = 0.54, p = 0.02). INTERPRETATION: Our results indicate that PLXNA4 has a role in AD pathogenesis through isoform-specific effects on tau phosphorylation

Increased degradation of FMRP contributes to neuronal hyperexcitability in tuberous sclerosis complex

Summary: Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental disorder, but new therapies have been impeded by a lack of understanding of the pathological mechanisms. Tuberous sclerosis complex (TSC) and fragile X syndrome are associated with alterations in the mechanistic target of rapamycin (mTOR) and fragile X messenger ribonucleoprotein 1 (FMRP), which have been implicated in the development of ASD. Previously, we observed that transcripts associated with FMRP were down-regulated in TSC2-deficient neurons. In this study, we find that FMRP turnover is dysregulated in TSC2-deficient rodent primary neurons and human induced pluripotent stem cell (iPSC)-derived neurons and is dependent on the E3 ubiquitin ligase anaphase-promoting complex. We also demonstrate that overexpression of FMRP can partially rescue hyperexcitability in TSC2-deficient iPSC-derived neurons. These data indicate that FMRP dysregulation represents an important pathological mechanism in the development of abnormal neuronal activity in TSC and illustrate a molecular convergence between these two neurogenetic disorders

Generation and characterization of six human induced pluripotent stem cell lines (hiPSCs) from three individuals with SSADH Deficiency and CRISPR-corrected isogenic controls

Succinic Semialdehyde Dehydrogenase Deficiency (SSADHD) is an ultra-rare autosomal recessive neurometabolic disorder caused by ALDH5A1 mutations presenting with autism and epilepsy. Here, we report the generation and characterization of human induced pluripotent stem cells (hiPSCs) derived from fibroblasts of three unrelated SSADHD patients – one female and two males with the CRISPR-corrected isogenic controls. These individuals are clinically diagnosed and are being followed in a longitudinal clinical study

Enhanced prime editing systems by manipulating cellular determinants of editing outcomes

While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types

MiR-142-3p regulates cortical oligodendrocyte gene co-expression networks associated with tauopathy

Oligodendrocytes exist in a heterogenous state and are implicated in multiple neuropsychiatric diseases including dementia. Cortical oligodendrocytes are a glial population uniquely positioned to play a key role in neurodegeneration by synchronizing circuit connectivity but molecular pathways specific to this role are lacking. We utilized oligodendrocyte-specific translating ribosome affinity purification and RNA-seq (TRAP-seq) to transcriptionally profile adult mature oligodendrocytes from different regions of the central nervous system. Weighted gene co-expression network analysis reveals distinct region-specific gene networks. Two of these mature myelinating oligodendrocyte gene networks uniquely define cortical oligodendrocytes and differentially regulate cortical myelination (M8) and synaptic signaling (M4). These two cortical oligodendrocyte gene networks are enriched for genes associated with dementia including MAPT and include multiple gene targets of the regulatory microRNA, miR-142-3p. Using a combination of TRAP-qPCR, miR-142-3p overexpression in vitro, and miR-142-null mice, we show that miR-142-3p negatively regulates cortical myelination. In rTg4510 tau-overexpressing mice, cortical myelination is compromised, and tau-mediated neurodegeneration is associated with gene co-expression networks that recapitulate both the M8 and M4 cortical oligodendrocyte gene networks identified from normal cortex. We further demonstrate overlapping gene networks in mature oligodendrocytes present in normal cortex, rTg4510 and miR-142-null mice, and existing datasets from human tauopathies to provide evidence for a critical role of miR-142-3p-regulated cortical myelination and oligodendrocyte-mediated synaptic signaling in neurodegeneration

miR-142-3p regulates cortical oligodendrocyte gene co-expression networks associated with tauopathy.

Oligodendrocytes exist in a heterogenous state and are implicated in multiple neuropsychiatric diseases including dementia. Cortical oligodendrocytes are a glial population uniquely positioned to play a key role in neurodegeneration by synchronizing circuit connectivity but molecular pathways specific to this role are lacking. We utilized oligodendrocyte-specific translating ribosome affinity purification and RNA-seq (TRAP-seq) to transcriptionally profile adult mature oligodendrocytes from different regions of the central nervous system. Weighted gene co-expression network analysis reveals distinct region-specific gene networks. Two of these mature myelinating oligodendrocyte gene networks uniquely define cortical oligodendrocytes and differentially regulate cortical myelination (M8) and synaptic signaling (M4). These two cortical oligodendrocyte gene networks are enriched for genes associated with dementia including MAPT and include multiple gene targets of the regulatory microRNA, miR-142-3p. Using a combination of TRAP-qPCR, miR-142-3p overexpression in vitro, and miR-142-null mice, we show that miR-142-3p negatively regulates cortical myelination. In rTg4510 tau-overexpressing mice, cortical myelination is compromised, and tau-mediated neurodegeneration is associated with gene co-expression networks that recapitulate both the M8 and M4 cortical oligodendrocyte gene networks identified from normal cortex. We further demonstrate overlapping gene networks in mature oligodendrocytes present in normal cortex, rTg4510 and miR-142-null mice, and existing datasets from human tauopathies to provide evidence for a critical role of miR-142-3p-regulated cortical myelination and oligodendrocyte-mediated synaptic signaling in neurodegeneration