Revisiting astrocyte to neuron conversion with lineage tracing in vivo.

In vivo cell fate conversions have emerged as potential regeneration-based therapeutics for injury and disease. Recent studies reported that ectopic expression or knockdown of certain factors can convert resident astrocytes into functional neurons with high efficiency, region specificity, and precise connectivity. However, using stringent lineage tracing in the mouse brain, we show that the presumed astrocyte-converted neurons are actually endogenous neurons. AAV-mediated co-expression of NEUROD1 and a reporter specifically and efficiently induces reporter-labeled neurons. However, these neurons cannot be traced retrospectively to quiescent or reactive astrocytes using lineage-mapping strategies. Instead, through a retrograde labeling approach, our results reveal that endogenous neurons are the source for these viral-reporter-labeled neurons. Similarly, despite efficient knockdown of PTBP1 in vivo, genetically traced resident astrocytes were not converted into neurons. Together, our results highlight the requirement of lineage-tracing strategies, which should be broadly applied to studies of cell fate conversions in vivo.

Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d.

CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function and explain its compact molecular architecture, we resolved cryoelectron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 Å resolution, respectively. Furthermore, a 6.5 Å reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, provide insights into its RNA-guided, RNA-targeting mechanism and delineate a blueprint for the rational design of improved transcriptome engineering technologies.

Defining a Cancer Dependency Map.

Most human epithelial tumors harbor numerous alterations, making it difficult to predict which genes are required for tumor survival. To systematically identify cancer dependencies, we analyzed 501 genome-scale loss-of-function screens performed in diverse human cancer cell lines. We developed DEMETER, an analytical framework that segregates on- from off-target effects of RNAi. 769 genes were differentially required in subsets of these cell lines at a threshold of six SDs from the mean. We found predictive models for 426 dependencies (55%) by nonlinear regression modeling considering 66,646 molecular features. Many dependencies fall into a limited number of classes, and unexpectedly, in 82% of models, the top biomarkers were expression based. We demonstrated the basis behind one such predictive model linking hypermethylation of the UBB ubiquitin gene to a dependency on UBC. Together, these observations provide a foundation for a cancer dependency map that facilitates the prioritization of therapeutic targets.

Apc Restoration Promotes Cellular Differentiation and Reestablishes Crypt Homeostasis in Colorectal Cancer.

The adenomatous polyposis coli (APC) tumor suppressor is mutated in the vast majority of human colorectal cancers (CRC) and leads to deregulated Wnt signaling. To determine whether Apc disruption is required for tumor maintenance, we developed a mouse model of CRC whereby Apc can be conditionally suppressed using a doxycycline-regulated shRNA. Apc suppression produces adenomas in both the small intestine and colon that, in the presence of Kras and p53 mutations, can progress to invasive carcinoma. In established tumors, Apc restoration drives rapid and widespread tumor-cell differentiation and sustained regression without relapse. Tumor regression is accompanied by the re-establishment of normal crypt-villus homeostasis, such that once aberrantly proliferating cells reacquire self-renewal and multi-lineage differentiation capability. Our study reveals that CRC cells can revert to functioning normal cells given appropriate signals and provide compelling in vivo validation of the Wnt pathway as a therapeutic target for treatment of CRC.

Therapeutic potential of RNA-enriched extracellular vesicles: The next generation in RNA delivery via biogenic nanoparticles.

Exosomes are extracellular vesicles (EVs) (∼50-150 nm) that have emerged as promising vehicles for therapeutic applications and drug delivery. These membrane-bound particles, released by all actively dividing cells, have the ability to transfer effector molecules, including proteins, RNA, and even DNA, from donor cells to recipient cells, thereby modulating cellular responses. RNA-based therapeutics, including microRNAs, messenger RNAs, long non-coding RNAs, and circular RNAs, hold great potential in controlling gene expression and treating a spectrum of medical conditions. RNAs encapsulated in EVs are protected from extracellular degradation, making them attractive for therapeutic applications. Understanding the intricate biology of cargo loading and transfer within EVs is pivotal to unlocking their therapeutic potential. This review discusses the biogenesis and classification of EVs, methods for loading RNA into EVs, their advantages as drug carriers over synthetic-lipid-based systems, and the potential applications in treating neurodegenerative diseases, cancer, and viral infections. Notably, EVs show promise in delivering RNA cargo across the blood-brain barrier and targeting tumor cells, offering a safe and effective approach to RNA-based therapy in these contexts.

Coupling shRNA screens with single-cell RNA-seq identifies a dual role for mTOR in reprogramming-induced senescence.

Expression of the transcription factors OCT4, SOX2, KLF4, and cMYC (OSKM) reprograms somatic cells into induced pluripotent stem cells (iPSCs). Reprogramming is a slow and inefficient process, suggesting the presence of safeguarding mechanisms that counteract cell fate conversion. One such mechanism is senescence. To identify modulators of reprogramming-induced senescence, we performed a genome-wide shRNA screen in primary human fibroblasts expressing OSKM. In the screen, we identified novel mediators of OSKM-induced senescence and validated previously implicated genes such as CDKN1A We developed an innovative approach that integrates single-cell RNA sequencing (scRNA-seq) with the shRNA screen to investigate the mechanism of action of the identified candidates. Our data unveiled regulation of senescence as a novel way by which mechanistic target of rapamycin (mTOR) influences reprogramming. On one hand, mTOR inhibition blunts the induction of cyclin-dependent kinase (CDK) inhibitors (CDKIs), including p16INK4a, p21CIP1, and p15INK4b, preventing OSKM-induced senescence. On the other hand, inhibition of mTOR blunts the senescence-associated secretory phenotype (SASP), which itself favors reprogramming. These contrasting actions contribute to explain the complex effect that mTOR has on reprogramming. Overall, our study highlights the advantage of combining functional screens with scRNA-seq to accelerate the discovery of pathways controlling complex phenotypes.

AAV mediated repression of Neat1 lncRNA combined with F8 gene augmentation mitigates pathological mediators of joint disease in haemophilia.

Haemophilic arthropathy (HA), a common comorbidity in haemophilic patients leads to joint pain, deformity and reduced quality of life. We have recently demonstrated that a long non-coding RNA, Neat1 as a primary regulator of matrix metalloproteinase (MMP) 3 and MMP13 activity, and its induction in the target joint has a deteriorating effect on articular cartilage. In the present study, we administered an Adeno-associated virus (AAV) 5 vector carrying an short hairpin (sh)RNA to Neat1 via intra-articular injection alone or in conjunction with systemic administration of a capsid-modified AAV8 (K31Q) vector carrying F8 gene (F8-BDD-V3) to study its impact on HA. AAV8K31Q-F8 vector administration at low dose, led to an increase in FVIII activity (16%-28%) in treated mice. We further observed a significant knockdown of Neat1 (~40 fold vs. untreated injured joint, p = 0.005) in joint tissue of treated mice and a downregulation of chondrodegenerative enzymes, MMP3, MMP13 and the inflammatory mediator- cPLA2, in mice receiving combination therapy. These data demonstrate that AAV mediated Neat1 knockdown in combination with F8 gene augmentation can potentially impact mediators of haemophilic joint disease.

Disruption of Golgi markers by two RILP-directed shRNAs in neurons: A new role for RILP or a neuron-specific off-target phenotype?

In neurons, degradation of dendritic cargos requires RAB7 and dynein-mediated retrograde transport to somatic lysosomes. To test if the dynein adapter RAB-interacting lysosomal protein (RILP) mediated the recruitment of dynein to late endosomes for retrograde transport in dendrites, we obtained several knockdown reagents previously validated in non-neuronal cells. Striking endosomal phenotypes elicited by one shRILP plasmid were not reproduced by another one. Furthermore, we discovered a profound depletion of Golgi/TGN markers for both shRILP plasmids. This Golgi disruption was only observed in neurons and could not be rescued by re-expression of RILP. This Golgi phenotype was also not found in neurons treated with siRILP or gRILP/Cas9. Lastly, we tested if a different RAB protein that interacts with RILP, namely the Golgi-associated RAB34, might be responsible for the loss of Golgi markers. Expression of a dominant-negative RAB34 did indeed cause changes in Golgi staining in a small subset of neurons but manifested as fragmentation rather than loss of staining. Unlike in non-neuronal cells, interference with RAB34 did not cause dispersal of lysosomes in neurons. Based on multiple lines of experimentation, we conclude that the neuronal Golgi phenotype observed with shRILP is likely off-target in this cell type specifically. Any observed disruptions of endosomal trafficking caused by shRILP in neurons might thus be downstream of Golgi disruption. It would be interesting to identify the actual target for this neuronal Golgi phenotype. Cell type-specific off-target phenotypes therefore likely occur in neurons, necessitating revalidation of reagents that were previously validated in other cell types.

Cdk5 inhibition in the SOD1G93A transgenic mouse model of amyotrophic lateral sclerosis suppresses neurodegeneration and extends survival.

Deregulated cyclin-dependent kinase 5 (Cdk5) activity closely correlates with hyperphosphorylated tau, a common pathology found in neurodegenerative diseases. Previous postmortem studies had revealed increased Cdk5 immunoreactivity in amyotrophic lateral sclerosis (ALS); hence, we investigated the effects of Cdk5 inhibition on ALS model mice and neurons in this study. For the in vitro study, motor neuron cell lines with wild-type superoxide dismutase 1 (SOD1) or SOD1G93A and primary neuronal cultures from SOD1G93A transgenic (TG) mice or non-TG mice were compared for the expression of proteins involved in tau pathology, neuroinflammation, apoptosis, and neuritic outgrowth by applying Cdk5-small interfering RNA or Cdk5-short hairpin RNA (shRNA). For the in vivo study, SOD1G93A mice and non-TG mice were intrathecally injected with adeno-associated virus 9 (AAV9)-scramble (SCR)-shRNA or AAV9-Cdk5-shRNA at the age of 5 weeks. Weight and motor function were measured three times per week from 60 days of age, longevity was evaluated, and the tissues were collected from 90-day-old or 120-day-old mice. Neurons with SOD1G93A showed increased phosphorylated tau, attenuated neuritic growth, mislocalization of SOD1, and enhanced neuroinflammation and apoptosis, all of which were reversed by Cdk5 inhibition. Weights did not show significant differences among non-TG and SOD1G93A mice with or without Cdk5 silencing. SOD1G93A mice treated with AAV9-Cdk5-shRNA showed significantly delayed disease onset, delayed rotarod failure, and prolonged survival compared with those treated with AAV9-SCR-shRNA. The brain and spinal cord of SOD1G93A mice intrathecally injected with AAV9-Cdk5-shRNA exhibited suppressed tau pathology, neuroinflammation, apoptosis, and an increased number of motor neurons compared to those of SOD1G93A mice injected with AAV9-SCR-shRNA. Cdk5 inhibition could be an important mechanism in the development of a new therapeutic strategy for ALS.

Dentate gyrus granule cells are a locus of pathology in Scn8a developmental encephalopathy.

Gain-of-function mutations in SCN8A cause developmental and epileptic encephalopathy (DEE), a disorder characterized by early-onset refractory seizures, deficits in motor and intellectual functions, and increased risk of sudden unexpected death in epilepsy. Altered activity of neurons in the corticohippocampal circuit has been reported in mouse models of DEE. We examined the effect of chronic seizures on gene expression in the hippocampus by single-nucleus RNA sequencing in mice expressing the patient mutation SCN8A-p.Asn1768Asp (N1768D). One hundred and eighty four differentially expressed genes were identified in dentate gyrus granule cells, many more than in other cell types. Electrophysiological recording from dentate gyrus granule cells demonstrated an elevated firing rate. Targeted reduction of Scn8a expression in the dentate gyrus by viral delivery of an shRNA resulted in doubling of median survival time from 4 months to 8 months, whereas delivery of shRNA to the CA1 and CA3 regions did not result in lengthened survival. These data indicate that granule cells of the dentate gyrus are a specific locus of pathology in SCN8A-DEE.

ILGBMSH: an interpretable classification model for the shRNA target prediction with ensemble learning algorithm.

Short hairpin RNA (shRNA)-mediated gene silencing is an important technology to achieve RNA interference, in which the design of potent and reliable shRNA molecules plays a crucial role. However, efficient shRNA target selection through biological technology is expensive and time consuming. Hence, it is crucial to develop a more precise and efficient computational method to design potent and reliable shRNA molecules. In this work, we present an interpretable classification model for the shRNA target prediction using the Light Gradient Boosting Machine algorithm called ILGBMSH. Rather than utilizing only the shRNA sequence feature, we extracted 554 biological and deep learning features, which were not considered in previous shRNA prediction research. We evaluated the performance of our model compared with the state-of-the-art shRNA target prediction models. Besides, we investigated the feature explanation from the model's parameters and interpretable method called Shapley Additive Explanations, which provided us with biological insights from the model. We used independent shRNA experiment data from other resources to prove the predictive ability and robustness of our model. Finally, we used our model to design the miR30-shRNA sequences and conducted a gene knockdown experiment. The experimental result was perfectly in correspondence with our expectation with a Pearson's coefficient correlation of 0.985. In summary, the ILGBMSH model can achieve state-of-the-art shRNA prediction performance and give biological insights from the machine learning model parameters.

miR-AB, a miRNA-based shRNA viral toolkit for multicolor-barcoded multiplex RNAi at a single-cell level.

Uncovering the functions of genes in a complex biological process is fundamental for systems biology. However, currently there is no simple and reliable experimental tool available to conduct loss-of-function experiments for multiple genes in every possible combination in a single experiment, which is vital for parsing the interactive role of multiple genes in a given phenotype. In this study, we develop miR-AB, a new microRNA-based shRNA (shRNAmir) backbone for simplified, cost-effective, and error-proof production of shRNAmirs. After verification of its potent RNAi efficiency in vitro and in vivo, miR-AB was integrated into a viral toolkit containing multiple eukaryotic promoters to enable its application in diverse cell types. We further engineer eight fluorescent proteins emitting wavelengths across the entire visible spectrum into this toolkit and use it to set up a multicolor-barcoded multiplex RNAi assay where multiple genes are strongly and reliably silenced both individually and combinatorially at a single-cell level.

RNAi screening reveals a synthetic chemical-genetic interaction between ATP synthase and PFK1 in cancer cells.

To meet cellular bioenergetic and biosynthetic demands, cancer cells remodel their metabolism to increase glycolytic flux, a phenomenon known as the Warburg effect and believed to contribute to cancer malignancy. Among glycolytic enzymes, phosphofructokinase-1 (PFK1) has been shown to act as a rate-limiting enzyme and to facilitate the Warburg effect in cancer cells. In this study, however, we found that decreased PFK1 activity did not affect cell survival or proliferation in cancer cells. This raised a question regarding the importance of PFK1 in malignancy. To gain insights into the role of PFK1 in cancer metabolism and the possibility of adopting it as a novel anticancer therapeutic target, we screened for genes that caused lethality when they were knocked down in the presence of tryptolinamide (TLAM), a PFK1 inhibitor. The screen revealed a synthetic chemical-genetic interaction between genes encoding subunits of ATP synthase (complex V) and TLAM. Indeed, after TLAM treatment, the sensitivity of HeLa cells to oligomycin A (OMA), an ATP synthase inhibitor, was 13,000 times higher than that of untreated cells. Furthermore, this sensitivity potentiation by TLAM treatment was recapitulated by genetic mutations of PFK1. By contrast, TLAM did not potentiate the sensitivity of normal fibroblast cell lines to OMA, possibly due to their reduced energy demands compared to cancer cells. We also showed that the PFK1-mediated glycolytic pathway can act as an energy reservoir. Selective potentiation of the efficacy of ATP synthase inhibitors by PFK1 inhibition may serve as a foundation for novel anticancer therapeutic strategies.

Induced regulatory T cells modified by knocking down T-bet in combination with ectopic expression of inhibitory cytokines effectively protect graft-versus-host disease.

Induced regulatory T (iTreg) cells play a vital role in immune tolerance and in controlling chronic inflammation. Generated in the periphery, iTreg cells are suitable for responding to alloantigens and preventing transplant rejection. Nevertheless, their clinical application has been impeded by the plasticity and instability attributed to the loss of forkhead box protein 3 expression, raising concerns that iTreg may be converted to effector T cells and even exert a pathogenic effect. Herein, second-generation short hairpin RNAs loaded with 3 pairs of small interfering RNAs were utilized to target the T-box transcription factor TBX21. In addition, 2 immunosuppressive cytokines, namely, transforming growth factor beta and interleukin 10, were constitutively expressed. This novel engineering strategy allowed the generation of stably induced regulatory T (SI Treg) cells, which maintained the expression of forkhead box protein 3 even in an unfavorable environment and exerted potent immunosuppressive functions in vitro. Furthermore, SI Treg cells demonstrated an effector transcriptional profile. Finally, SI Treg cells showed a significant protective effect against graft-versus-host disease-related deaths in a xenotransplantation model. Collectively, these results signify that SI Treg cells hold great promise for future clinical application and offer a rational therapeutic approach for transplant rejection.

Development of short hairpin RNA expression vectors targeting the internal ribosomal entry site of the classical swine fever virus genomic RNA.

BACKGROUND: Classical swine fever (CSF) is a fatal contagious disease affecting pigs caused by classical swine fever virus (CSFV). The disease can be transmitted by pigs and wild boars, and it is difficult to prevent and control. To obtain necessary information to establish the CSFV resistant animals in a future study, we designed lentiviral vector-delivered short hairpin RNAs (shRNAs) targeting the conserved domain III of the internal ribosomal entry site (IRES) of the CSFV genomic RNA. RESULTS: First, we confirmed the effects of siRNAs on CSFV-IRES activity. We observed significant inhibition of CSFV-IRES activity by si42 (domain IIIa), si107 (domain IIIc), and si198 (domain IIIf) in SK-L cells and si56 (domain IIIb), si142 (domain IIId1) and si198 in HEK293 cells without affecting the amount of luciferase RNA. Next, we constructed lentiviral vectors expressing shRNA based on siRNA sequences. Treatment with shRNA-expressing lentivirus was examined at 7 and 14 days post infection in SK-L cells and HEK293 cells, and CSFV-IRES was significantly suppressed at 14 days (sh42) post infection in HEK293 cells without significant cytotoxicity. Next, we examined the silencing effect of siRNA on CSFV replicon RNA and observed a significant effect by si198 after 2 days of treatment and by shRNA-expressing lentivirus (sh56, sh142, and sh198) infection after 14 days of treatment. Treatment of sh198-expressing lentivirus significantly suppressed CSFV infection at 3 days after infection. CONCLUSION: The IRES targeting sh198 expressing lentivirus vector can be a candidate tool for CSFV infection control.

Differential Roles of Oxytocin Receptors in the Prefrontal Cortex and Nucleus Accumbens on Cocaine Self-Administration and Reinstatement of Cued Cocaine Seeking in Male Rats.

BACKGROUND: Little is known about the specific roles of cortical and accumbal oxytocin receptors in drug use disorders. To better understand the importance of the endogenous oxytocin system in cocaine relapse behavior, we developed an adeno-associated viral vector-expressing short hairpin (sh) RNAs to selectively degrade the rat oxytocin receptor (OxyR) mRNA in vivo. METHODS: Male (Sprague-Dawley) rats received bilateral infusions of the shRNA for the oxytocin receptor (shOxyR) or an shRNA control virus into the prefrontal cortex (PFC) or the nucleus accumbens core (NAc). Rats self-administered cocaine on an escalating FR ratio for 14 days, lever responding was extinguished, and rats were tested for cued and cocaine-primed reinstatement of drug seeking. RESULTS: OxyR knockdown in the PFC delayed the acquisition of lever pressing on an fixed ratio 1 schedule of reinforcement. All rats eventually acquired the same level of lever pressing and discrimination, and there were no differences in extinction. OxyR knockdown in the NAc had no effect during acquisition. In both the PFC and NAc, the shOxyR decreased cued reinstatement relative to shRNA control virus but was without effect during drug-primed reinstatement. OxyR knockdown in the PFC increased chamber activity during a social interaction task. CONCLUSIONS: This study provides critical new information about how endogenous OxyRs function to affect drug seeking in response to different precipitators of relapse. The tool developed to knockdown OxyRs in rat could provide important new insights that aid development of oxytocin-based therapeutics to reduce return-to-use episodes in people with substance use disorder and other neuropsychiatric disorders.

Inefficient uptake of small interfering RNAs is responsible for their inability to trigger RNA interference in Colorado potato beetle cells.

There has been limited success in the usage of exogenous small interference RNA (siRNA) or small hairpin RNA (shRNA) to trigger RNA interference (RNAi) in insects. Instead, long double-stranded RNAs (dsRNA) are used to induce knockdown of target genes in insects. Here, we compared the potency of si/sh RNAs and dsRNA in Colorado potato beetle (CPB) cells. CPB cells showed highly efficient RNAi response to dsRNA. However, si/sh RNAs were inefficient in triggering RNAi in CPB cells. Confocal microscopy observations of Cy3 labeled-si/sh RNA cellular uptake revealed reduced si/sh RNA uptake compared to dsRNA. si/sh RNAs were stable in the conditioned media of CPB cells. Although in a small amount, when internalized by CPB cells, the si/sh RNAs were processed by the Dicer enzyme. Lipid-mediated transfection and chimeric dsRNA approaches were used to improve the delivery of si/sh RNAs. Our results suggest that the uptake of si/sh RNAs is inefficient in CPB cells, resulting in ineffective RNAi response. However, with the help of effective delivery methods, si/sh RNA could be a useful option for developing target-specific RNAi-mediated biopesticides.

PD-1 and TIGIT downregulation distinctly affect the effector and early memory phenotypes of CD19-targeting CAR T cells.

CD19-targeting chimeric antigen receptor (CAR) T cells have become an important therapeutic option for patients with relapsed and refractory B cell malignancies. However, a significant portion of patients still do not benefit from the therapy owing to various resistance mechanisms, including high expression of multiple inhibitory immune checkpoint receptors. Here, we report a lentiviral two-in-one CAR T approach in which two checkpoint receptors are downregulated simultaneously by a dual short hairpin RNA cassette integrated into a CAR vector. Using this system, we evaluated CD19-targeting CAR T cells in the context of four different checkpoint combinations-PD-1/TIM-3, PD-1/LAG-3, PD-1/CTLA-4, and PD-1/TIGIT-and found that CAR T cells with PD-1/TIGIT downregulation uniquely exerted synergistic antitumor effects. Importantly, functional and phenotypic analyses suggested that downregulation of PD-1 enhances short-term effector function, whereas downregulation of TIGIT is primarily responsible for maintaining a less differentiated/exhausted state, providing a potential mechanism for the observed synergy. The PD-1/TIGIT-downregulated CAR T cells generated from diffuse large B cell lymphoma patient-derived T cells also showed robust antitumor activity and significantly improved persistence in vivo. The efficacy and safety of PD-1/TIGIT-downregulated CD19-targeting CAR T cells are currently being evaluated in adult patients with relapsed or refractory large B cell lymphoma (ClinicalTrials.gov: NCT04836507).

Direct screening for effective shRNA with a single mismatch in human cells without laborious cloning.

We have devised a method for the direct screening of efficient short hairpin (sh)RNA molecules in human cells, eliminating the need for the time-consuming process of cloning in Escherichia coli. Our screening suggested that single mismatches to shRNAs can significantly alter their activity.

IRS1 expression in hippocampus is age-dependent and is required for mature spine maintenance and neuritogenesis.

Insulin and insulin-like growth factor type I (IGF-1) play prominent roles in brain activity throughout the lifespan. Insulin/IGF1 signaling starts with the activation of the intracellular insulin receptor substrates (IRS). In this work, we performed a comparative study of IRS1 and IRS2, together with the IGF1 (IGF1R) and insulin (IR) receptor expression in the hippocampus and prefrontal cortex during development. We found that IRS1 and IRS2 expression is prominent during development and declines in the aged hippocampus, contrary to IR, which increases in adulthood and aging. In contrast, IGF1R expression is unaffected by age. Expression patterns are similar in the prefrontal cortex. Neurite development occurs postnatally in the rodent hippocampus and cortex, and it declines in the mature and aged brain and is influenced by trophic factors. In our previous work, we demonstrated that knockdown of IRS1 by shRNA impairs learning and reduces synaptic plasticity in a rat model, as measured by synaptophysin puncta in axons. In this study, we report that shIRS1 alters spine maturation in adult hilar hippocampal neurons. Lastly, to understand the role of IRS1 in neuronal neurite tree, we transfect shIRS1 into primary neuronal cultures and observed that shIRS1 reduced neurite branching and neurite length. Our results demonstrate that IRS1/2 and insulin/IGF1 receptors display different age-dependent expression profiles and that IRS1 is required for spine maturation, demonstrating a novel role for IRS1 in synaptic plasticity.