Sterile inflammation via TRPM8 RNA-dependent TLR3-NF-kB/IRF3 activation promotes antitumor immunity in prostate cancer.

Inflammation is a common condition of prostate tissue, whose impact on carcinogenesis is highly debated. Microbial colonization is a well-documented cause of a small percentage of prostatitis cases, but it remains unclear what underlies the majority of sterile inflammation reported. Here, androgen- independent fluctuations of PSA expression in prostate cells have lead us to identify a prominent function of the Transient Receptor Potential Cation Channel Subfamily M Member 8 (TRPM8) gene in sterile inflammation. Prostate cells secret TRPM8 RNA into extracellular vesicles (EVs), which primes TLR3/NF-kB-mediated inflammatory signaling after EV endocytosis by epithelial cancer cells. Furthermore, prostate cancer xenografts expressing a translation-defective form of TRPM8 RNA contain less collagen type I in the extracellular matrix, significantly more infiltrating NK cells, and larger necrotic areas as compared to control xenografts. These findings imply sustained, androgen-independent expression of TRPM8 constitutes as a promoter of anticancer innate immunity, which may constitute a clinically relevant condition affecting prostate cancer prognosis.

Intra-epithelial non-canonical Activin A signaling safeguards prostate progenitor quiescence.

The healthy prostate is a relatively quiescent tissue. Yet, prostate epithelium overgrowth is a common condition during aging, associated with urinary dysfunction and tumorigenesis. For over thirty years, TGF-ß ligands have been known to induce cytostasis in a variety of epithelia, but the intracellular pathway mediating this signal in the prostate, and its relevance for quiescence, have remained elusive. Here, using mouse prostate organoids to model epithelial progenitors, we find that intra-epithelial non-canonical Activin A signaling inhibits cell proliferation in a Smad-independent manner. Mechanistically, Activin A triggers Tak1 and p38 ΜAPK activity, leading to p16 and p21 nuclear import. Spontaneous evasion from this quiescent state occurs upon prolonged culture, due to reduced Activin A secretion, a condition associated with DNA replication stress and aneuploidy. Organoids capable to escape quiescence in vitro are also able to implant with increased frequency into immunocompetent mice. This study demonstrates that non-canonical Activin A signaling safeguards epithelial quiescence in the healthy prostate, with potential implications for the understanding of cancer initiation, and the development of therapies targeting quiescent tumor progenitors.

TRPM8 protein expression in hormone naïve local and lymph node metastatic prostate cancer.

OBJECTIVE: Prostate cancer (PCa) is the second most common malignancy in men. Radiotherapy and surgery successfully control organ-confined tumors, although, locally advanced/high-risk PCa frequently progress to the metastatic stage of the disease, which is uncurable. Identification of novel strategies to improve the efficacy of standard clinical protocols is a primary need. Among the molecular targets of potential clinical interest recently highlighted by accurate preclinical studies, the TRPM8 cation channel is particularly promising. In this study, we aim at establishing a standardized immunohistochemistry protocol to evaluate TRPM8 expression in normal and pathological prostate tissues. METHODS: The specificity and sensitivity of TRPM8 antibody ACC-049 was validated in different human prostate cell lines by western blot and immunocytochemistry analyses. Expression of the TRPM8 channel in normal and pathological prostate tissue was evaluated by immunohistochemistry using a tissue microarray containing 58 cases of prostate adenocarcinomas and in primary and lymph nodes metastatic human PCa matched specimens. RESULTS: TRPM8 expression marks luminal epithelial cells in benign prostate tissue. In malignant lesions of the prostate, TRPM8 expression is frequently more abundant in advanced stages of the disease (PCa stage III/IV). Finally, lymph node metastases and matched primary tumors show similar amounts of the channel. CONCLUSIONS: Collectively, our results reinforce the importance of TRPM8 as prostate biomarker and emphasize the value of the channel as promising novel molecular target for the treatment of prostate adenocarcinoma.

The Chemokine CCL2 Mediates the Seizure-enhancing Effects of Systemic Inflammation.

Epilepsy is a chronic disorder characterized by spontaneous recurrent seizures. Brain inflammation is increasingly recognized as a critical factor for seizure precipitation, but the molecular mediators of such proconvulsant effects are only partly understood. The chemokine CCL2 is one of the most elevated inflammatory mediators in patients with pharmacoresistent epilepsy, but its contribution to seizure generation remains unexplored. Here, we show, for the first time, a crucial role for CCL2 and its receptor CCR2 in seizure control. We imposed a systemic inflammatory challenge via lipopolysaccharide (LPS) administration in mice with mesial temporal lobe epilepsy. We found that LPS dramatically increased seizure frequency and upregulated the expression of many inflammatory proteins, including CCL2. To test the proconvulsant role of CCL2, we administered systemically either a CCL2 transcription inhibitor (bindarit) or a selective antagonist of the CCR2 receptor (RS102895). We found that interference with CCL2 signaling potently suppressed LPS-induced seizures. Intracerebral administration of anti-CCL2 antibodies also abrogated LPS-mediated seizure enhancement in chronically epileptic animals. Our results reveal that CCL2 is a key mediator in the molecular pathways that link peripheral inflammation with neuronal hyperexcitability. SIGNIFICANCE STATEMENT: Substantial evidence points to a role for inflammation in epilepsy, but currently there is little insight as to how inflammatory pathways impact on seizure generation. Here, we examine the molecular mediators linking peripheral inflammation with seizure susceptibility in mice with mesial temporal lobe epilepsy. We show that a systemic inflammatory challenge via lipopolysaccharide administration potently enhances seizure frequency and upregulates the expression of the chemokine CCL2. Remarkably, selective pharmacological interference with CCL2 or its receptor CCR2 suppresses lipopolysaccharide-induced seizure enhancement. Thus, CCL2/CCR2 signaling plays a key role in linking systemic inflammation with seizure susceptibility.

Hippocampal dysregulation of neurofibromin-dependent pathways is associated with impaired spatial learning in engrailed 2 knock-out mice.

Genome-wide association studies indicated the homeobox-containing transcription factor Engrailed-2 (En2) as a candidate gene for autism spectrum disorders (ASD). Accordingly, En2 knock-out (En2(-/-)) mice show anatomical and behavioral "ASD-like" features, including decreased sociability and learning deficits. The molecular pathways underlying these deficits in En2(-/-) mice are not known. Deficits in signaling pathways involving neurofibromin and extracellular-regulated kinase (ERK) have been associated with impaired learning. Here we investigated the neurofibromin-ERK cascade in the hippocampus of wild-type (WT) and En2(-/-) mice before and after spatial learning testing. When compared with WT littermates, En2(-/-) mice showed impaired performance in the Morris water maze (MWM), which was accompanied by lower expression of the activity-dependent gene Arc. Quantitative RT-PCR, immunoblotting, and immunohistochemistry experiments showed a marked downregulation of neurofibromin expression in the dentate gyrus of both naive and MWM-treated En2(-/-) mice. ERK phosphorylation, known to be induced in the presence of neurofibromin deficiency, was increased in the dentate gyrus of En2(-/-) mice after MWM. Treatment of En2(-/-) mice with lovastatin, an indirect inhibitor of ERK phosphorylation, markedly reduced ERK phosphorylation in the dentate gyrus, but was unable to rescue learning deficits in MWM-trained mutant mice. Further investigation is needed to unravel the complex molecular mechanisms linking dysregulation of neurofibromin-dependent pathways to spatial learning deficits in the En2 mouse model of ASD.

Rarγ -Foxa1 signaling promotes luminal identity in prostate progenitors and is disrupted in prostate cancer.

Retinoic acid (RA) signaling is a master regulator of vertebrate development with crucial roles in directing body axis orientation and tissue differentiation, including in the reproductive system. However, a mechanistic understanding of how RA signaling promotes cell lineage identity in different tissues is often missing. Here, leveraging prostate organoid technology, we demonstrated that RA signaling orchestrates the commitment of adult mouse prostate progenitors to glandular identity, epithelial barrier integrity, and ultimately, proper specification of the prostatic lumen. Mechanistically, RA-dependent RARγ activation promotes the expression of the pioneer factor Foxa1, which synergizes with the androgen pathway for proper luminal expansion, cytoarchitecture and function. FOXA1 nucleotide variants are common in human prostate and breast cancers and considered driver mutations, though their pathogenic mechanism is incompletely understood. Combining functional genetics experiments with structural modeling of FOXA1 folding and chromatin binding analyses, we discovered that FOXA1 F254E255 is a loss-of-function mutation leading to compromised transcriptional function and lack of luminal fate commitment of prostate progenitors. Overall, we define RA as a crucial instructive signal for glandular identity in adult prostate progenitors. We propose deregulation of vitamin A metabolism as a risk factor for benign and malignant prostate disease, and identified cancer associated FOXA1 indels affecting residue F254 as loss-of-function mutations promoting dedifferentiation of adult prostate progenitors. Summary: Retinoic acid signaling orchestrates luminal differentiation of adult prostate progenitors.

Down-regulation of Shadoo in prion infections traces a pre-clinical event inversely related to PrP(Sc) accumulation.

During prion infections of the central nervous system (CNS) the cellular prion protein, PrP(C), is templated to a conformationally distinct form, PrP(Sc). Recent studies have demonstrated that the Sprn gene encodes a GPI-linked glycoprotein Shadoo (Sho), which localizes to a similar membrane environment as PrP(C) and is reduced in the brains of rodents with terminal prion disease. Here, analyses of prion-infected mice revealed that down-regulation of Sho protein was not related to Sprn mRNA abundance at any stage in prion infection. Down-regulation was robust upon propagation of a variety of prion strains in Prnp(a) and Prnp(b) mice, with the exception of the mouse-adapted BSE strain 301 V. In addition, Sho encoded by a TgSprn transgene was down-regulated to the same extent as endogenous Sho. Reduced Sho levels were not seen in a tauopathy, in chemically induced spongiform degeneration or in transgenic mice expressing the extracellular ADan amyloid peptide of familial Danish dementia. Insofar as prion-infected Prnp hemizygous mice exhibited accumulation of PrP(Sc) and down-regulation of Sho hundreds of days prior to onset of neurologic symptoms, Sho depletion can be excluded as an important trigger for clinical disease or as a simple consequence of neuronal damage. These studies instead define a disease-specific effect, and we hypothesize that membrane-associated Sho comprises a bystander substrate for processes degrading PrP(Sc). Thus, while protease-resistant PrP detected by in vitro digestion allows post mortem diagnosis, decreased levels of endogenous Sho may trace an early response to PrP(Sc) accumulation that operates in the CNS in vivo. This cellular response may offer new insights into the homeostatic mechanisms involved in detection and clearance of the misfolded proteins that drive prion disease pathogenesis.

RGG repeats of PrP-like Shadoo protein bind nucleic acids.

Shadoo (Sho) is a central nervous system glycoprotein with characteristics similar to those of the cellular prion protein PrP(C), each containing a highly conserved hydrophobic domain (HD) and an N-terminal repeat region. Whereas PrP(C) includes histidine-containing octarepeats, the Sho region N-terminal to the HD includes tandem positively charged "RGG boxes", predicted to bind RNA. Here, we demonstrate that Sho binds DNA and RNA in vitro via this arginine-rich region.

Immune dysfunction in the cerebellum of mice lacking the autism candidate gene Engrailed 2.

Immune system dysfunction has been described in autism spectrum disorder. Here we tested the hypothesis that cerebellar defects are accompanied by immune dysfunction in adult mice lacking the autism-candidate gene Engrailed 2 (En2). Gene ontology analyses revealed that biological processes related to immune function were over-represented in the cerebellar transcriptome of En2-/- mice. Pro-inflammatory molecules and chemokines were reduced in the En2-/- cerebellum compared to controls. Conversely, pro-inflammatory molecules were increased in the peripheral blood of mutant mice. Our results suggest a link between immune dysfunction and cerebellar defects detected in En2-/- mice.

Trpm8 Expression in Human and Mouse Castration Resistant Prostate Adenocarcinoma Paves the Way for the Preclinical Development of TRPM8-Based Targeted Therapies.

Metastatic prostate cancer (mPCa) is one of the leading causes of cancer-related mortality in both the US and Europe. Androgen deprivation is the first-line therapy for mPCa; however, resistance to therapy inevitably occurs and the disease progresses to the castration resistant stage, which is uncurable. A definition of novel targeted therapies is necessary for the establishment of innovative and more effective protocols of personalized oncology. We employed genetically engineered mouse models of PCa and human samples to characterize the expression of the TRPM8 cation channel in both hormone naïve and castration resistant tumors. We show that Trpm8 expression marks both indolent (Pten-null) and aggressive (Pten/Trp53 double-null and TRAMP) mouse prostate adenocarcinomas. Importantly, both mouse and human castration-resistant PCa preserve TRPM8 protein expression. Finally, we tested the effect of TRPM8 agonist D-3263 administration in combination with enzalutamide or docetaxel on the viability of aggressive mouse PCa cell lines. Our data demonstrate that D-3263 substantially enhances the pro-apoptotic activity of enzalutamide and docetaxel in TRAMP-C1 e TRAMP-C2 PCa cell lines. To conclude, this study provides the basis for pre-clinical in vivo testing of TRPM8 targeting as a novel strategy to implement the efficacy of standard-of-care treatments for advanced PCa.

ETS-related gene (ERG) undermines genome stability in mouse prostate progenitors via Gsk3ß dependent Nkx3.1 degradation.

21q22.2-3 deletion is the most common copy number alteration in prostate cancer (PCa). The genomic rearrangement results in the androgen-dependent de novo expression of ETS-related gene (ERG) in prostate cancer cells, a condition promoting tumor progression to advanced stages of the disease. Interestingly, ERG expression characterizes 5-30% of tumor precursor lesions - High Grade Prostatic Intraepithelial Neoplasia (HGPIN) - where its role remains unclear. Here, by combining organoids technology with Click-chemistry coupled Mass Spectrometry, we demonstrate a prominent role of ERG in remodeling the protein secretome of prostate progenitors. Functionally, by lowering autocrine Wnt-4 signaling, ERG represses canonical Wnt pathway in prostate progenitors, and, in turn, promotes the accumulation of DNA double strand breaks via Gsk3ß-dependent degradation of the tumor suppressor Nkx3.1. On the other hand, by shaping extracellular paracrine signals, ERG strengthens the pro-oxidative transcriptional signature of inflammatory macrophages, which we demonstrate to infiltrate pre-malignant ERG positive prostate lesions. These findings highlight previously unrecognized functions of ERG in undermining adult prostate progenitor niche through cell autonomous and non-autonomous mechanisms. Overall, by supporting the survival and proliferation of prostate progenitors in the absence of growth stimuli and promoting the accumulation of DNA damage through destabilization of Nkx3.1, ERG could orchestrate the prelude to neoplastic transformation.

Tune the channel: TRPM8 targeting in prostate cancer.

The therapeutic landscape of cancer treatments is quickly evolving thanks to the advent of precision oncology. Discovery of novel druggable targets and more reliable biomarkers is a primary objective towards personalized strategies of cancer treatment. Highly expressed in the prostate epithelium within the human body, Transient Receptor Potential subfamily M member 8 (TRPM8) levels rise in primary and hormone naïve metastatic prostate cancer (PCa) lesions, which makes this channel an interesting prototype of molecular target. Recently, by combining a multidisciplinary approach to an in vitro genetic platform, we demonstrated that the combination of potent TRPM8 agonists with X-rays induces a massive apoptotic response in radioresistant pre-malignant and malignant models of primary prostate lesions. As well, TRPM8 activation enhances the efficacy of docetaxel or enzalutamide in eradicating hormone naïve metastatic PCa cells. Overall, our findings provide a solid rationale for pursuing the pre-clinical and clinical study of TRPM8 as a valuable target for future approaches of precise oncology in PCa.

Wild-type Shadoo proteins convert to amyloid-like forms under native conditions.

The cellular prion protein PrP(C) refolds into a beta-sheet enriched, infectivity-associated form called PrP(Sc). Shadoo (Sho) is a newly discovered glycoprotein that is also expressed in the adult brain. Wild type (wt) mouse Sho consists of an arginine-rich region, a hydrophobic central domain of five tandem A/LAAG amino acid repeats R1-R5 with similarity to the hydrophobic domain of PrP(C), and a C-terminal domain with one N-linked carbohydrate. As some alanine-rich proteins and PrP with a shortened C-terminal domain form amyloid we investigated conformational properties of wt Sho and polymorphic variants with insertion/deletions centered on R3. Recombinant mouse and sheep Sho converted to an amyloid-like form without recourse to chemical denaturation or acidification. For wt proteins this transition was marked by increased thioflavin T binding, Congo red staining, presence of fibrillar structures by electron microscopy, formation of sodium dodecyl sulfate-resistant complexes and the generation of a C-terminal proteinase K resistant core of 5-8 kDa. Variant Sho proteins differing within the R1-R5 region exhibited most but not all of these properties. Our studies define a proteinase K -resistant signature fragment for the amyloid fold of Sho and raise the question of a physiological role for this form of the wt protein.

The Organoid Era Permits the Development of New Applications to Study Glioblastoma.

Glioblastoma (GB) is the most frequent and aggressive type of glioma. The lack of reliable GB models, together with its considerable clinical heterogeneity, has impaired a comprehensive investigation of the mechanisms that lead to tumorigenesis, cancer progression, and response to treatments. Recently, 3D cultures have opened the possibility to overcome these challenges and cerebral organoids are emerging as a leading-edge tool in GB research. The opportunity to easily engineer brain organoids via gene editing and to perform co-cultures with patient-derived tumor spheroids has enabled the analysis of cancer development in a context that better mimics brain tissue architecture. Moreover, the establishment of biobanks from GB patient-derived organoids represents a crucial starting point to improve precision medicine therapies. This review exemplifies relevant aspects of 3D models of glioblastoma, with a specific focus on organoids and their involvement in basic and translational research.

Calcium cytotoxicity sensitizes prostate cancer cells to standard-of-care treatments for locally advanced tumors.

Therapy resistance is a major roadblock in oncology. Exacerbation of molecular dysfunctions typical of cancer cells have proven effective in twisting oncogenic mechanisms to lethal conditions, thus offering new therapeutic avenues for cancer treatment. Here, we demonstrate that selective agonists of Transient Receptor Potential cation channel subfamily M member 8 (TRPM8), a cation channel characteristic of the prostate epithelium frequently overexpressed in advanced stage III/IV prostate cancers (PCa), sensitize therapy refractory models of PCa to radio, chemo or hormonal treatment. Overall, our study demonstrates that pharmacological-induced Ca2+ cytotoxicity is an actionable strategy to sensitize cancer cells to standard therapies.

Hippocampal dysregulation of FMRP/mGluR5 signaling in engrailed-2 knockout mice: a model of autism spectrum disorders.

Many evidences indicate that mice lacking the homeobox transcription factor engrailed-2 (En2(-/-) mice) represent a reliable model to investigate neurodevelopmental basis and gene expression changes relevant to autism spectrum disorders. Dysfunctions in fragile X mental retardation protein (FMRP), metabotropic glutamate receptor 5 (mGluR5), and GABAergic signaling pathways have been proposed as a possible pathogenic mechanism of autism spectrum disorders. Here, we exploited En2(-/-) mice to investigate hippocampal expression of FMRP, mGluR5, and GABA(A) receptor ß3 subunit (GABRB3). Quantitative reverse-transcription PCR showed that all these mRNAs were significantly downregulated in En2(-/-) mice compared with wild-type littermates. Western blot and immunohistochemistry confirmed the downregulation of FMRP and GABRB3 proteins, while showing a significant increase of mGluR5 protein in the En2(-/-) hippocampus. Our results suggest that the dysregulation of FMRP-mGluR5 signaling pathway, accompanied with a downregulation of GABRB3 expression, may contribute to the 'autistic-like' features observed in En2 mice, providing possible molecular targets for future pharmacological studies.

Human Doppel and prion protein share common membrane microdomains and internalization pathways.

Doppel is the first identified homologue of the prion protein (PrPc) implicated in prion disease. Doppel is considered an N-truncated form of PrPc, and shares with PrPc several structural and biochemical features. When over expressed in the brain of some PrP knockout animals, it provokes cerebellar ataxia. As this phenotype is rescued by reintroducing the PrP gene, it has been suggested that Doppel and PrPc have antagonistic functions and may compete for a common ligand. However, a direct interaction between the two proteins has recently been observed. To investigate whether the neuronal environment is suitable for such possibility, human Doppel and PrPc were expressed separately, or together, in neuroblastoma cells, and then studied by biochemical and immunomicroscopic tools, as well as in intact cells expressing fluorescent fusion constructs. The results demonstrate that Doppel and PrPc co-patch extensively at the plasma membrane, and get internalized together after ganglioside cross-linking by cholera toxin or addition of an antibody against only one of the proteins. These processes no longer occur if the integrity of rafts is disrupted. We also show that, whereas each protein expressed alone occupies Triton X-100-insoluble membrane microdomains, co-transfected Doppel and PrPc redistribute together into a less ordered lipidic environment. All these features are consistent with interactions occurring between Doppel and PrPc in our neuronal cell model.

GH Dysfunction in Engrailed-2 Knockout Mice, a Model for Autism Spectrum Disorders.

Insulin-like growth factor 1 (IGF-1) signaling promotes brain development and plasticity. Altered IGF-1 expression has been associated to autism spectrum disorders (ASD). IGF-1 levels were found increased in the blood and decreased in the cerebrospinal fluid of ASD children. Accordingly, IGF-1 treatment can rescue behavioral deficits in mouse models of ASD, and IGF-1 trials have been proposed for ASD children. IGF-1 is mainly synthesized in the liver, and its synthesis is dependent on growth hormone (GH) produced in the pituitary gland. GH also modulates cognitive functions, and altered levels of GH have been detected in ASD patients. Here, we analyzed the expression of GH, IGF-1, their receptors, and regulatory hormones in the neuroendocrine system of adult male mice lacking the homeobox transcription factor Engrailed-2 (En2 (-/-) mice). En2 (-/-) mice display ASD-like behaviors (social interactions, defective spatial learning, increased seizure susceptibility) accompanied by relevant neuropathological changes (loss of cerebellar and forebrain inhibitory neurons). Recent studies showed that En2 modulates IGF-1 activity during postnatal cerebellar development. We found that GH mRNA expression was markedly deregulated throughout the neuroendocrine axis in En2 (-/-) mice, as compared to wild-type controls. In mutant mice, GH mRNA levels were significantly increased in the pituitary gland, blood, and liver, whereas decreased levels were detected in the hippocampus. These changes were paralleled by decreased levels of GH protein in the hippocampus but not other tissues of En2 (-/-) mice. IGF-1 mRNA was significantly up-regulated in the liver and down-regulated in the En2 (-/-) hippocampus, but no differences were detected in the levels of IGF-1 protein between the two genotypes. Our data strengthen the notion that altered GH levels in the hippocampus may be involved in learning disabilities associated to ASD.

Altered GABAergic markers, increased binocularity and reduced plasticity in the visual cortex of Engrailed-2 knockout mice.

The maturation of the GABAergic system is a crucial determinant of cortical development during early postnatal life, when sensory circuits undergo a process of activity-dependent refinement. An altered excitatory/inhibitory balance has been proposed as a possible pathogenic mechanism of autism spectrum disorders (ASD). The homeobox-containing transcription factor Engrailed-2 (En2) has been associated to ASD, and En2 knockout (En2 (-/-)) mice show ASD-like features accompanied by a partial loss of cortical GABAergic interneurons. Here we studied GABAergic markers and cortical function in En2 (-/-) mice, by exploiting the well-known anatomical and functional features of the mouse visual system. En2 is expressed in the visual cortex at postnatal day 30 and during adulthood. When compared to age-matched En2 (+/+) controls, En2 (-/-) mice showed an increased number of parvalbumin (PV(+)), somatostatin (SOM(+)), and neuropeptide Y (NPY(+)) positive interneurons in the visual cortex at P30, and a decreased number of SOM(+) and NPY(+) interneurons in the adult. At both ages, the differences in distinct interneuron populations observed between En2 (+/+) and En2 (-/-) mice were layer-specific. Adult En2 (-/-) mice displayed a normal eye-specific segregation in the retino-geniculate pathway, and in vivo electrophysiological recordings showed a normal development of basic functional properties (acuity, response latency, receptive field size) of the En2 (-/-) primary visual cortex. However, a significant increase of binocularity was found in P30 and adult En2 (-/-) mice, as compared to age-matched controls. Differently from what observed in En2 (+/+) mice, the En2 (-/-) primary visual cortex did not respond to a brief monocular deprivation performed between P26 and P29, during the so-called "critical period." These data suggest that altered GABAergic circuits impact baseline binocularity and plasticity in En2 (-/-) mice, while leaving other visual functional properties unaffected.

Transcriptome profiling in engrailed-2 mutant mice reveals common molecular pathways associated with autism spectrum disorders.

BACKGROUND: Transcriptome analysis has been used in autism spectrum disorder (ASD) to unravel common pathogenic pathways based on the assumption that distinct rare genetic variants or epigenetic modifications affect common biological pathways. To unravel recurrent ASD-related neuropathological mechanisms, we took advantage of the En2-/- mouse model and performed transcriptome profiling on cerebellar and hippocampal adult tissues. METHODS: Cerebellar and hippocampal tissue samples from three En2-/- and wild type (WT) littermate mice were assessed for differential gene expression using microarray hybridization followed by RankProd analysis. To identify functional categories overrepresented in the differentially expressed genes, we used integrated gene-network analysis, gene ontology enrichment and mouse phenotype ontology analysis. Furthermore, we performed direct enrichment analysis of ASD-associated genes from the SFARI repository in our differentially expressed genes. RESULTS: Given the limited number of animals used in the study, we used permissive criteria and identified 842 differentially expressed genes in En2-/- cerebellum and 862 in the En2-/- hippocampus. Our functional analysis revealed that the molecular signature of En2-/- cerebellum and hippocampus shares convergent pathological pathways with ASD, including abnormal synaptic transmission, altered developmental processes and increased immune response. Furthermore, when directly compared to the repository of the SFARI database, our differentially expressed genes in the hippocampus showed enrichment of ASD-associated genes significantly higher than previously reported. qPCR was performed for representative genes to confirm relative transcript levels compared to those detected in microarrays. CONCLUSIONS: Despite the limited number of animals used in the study, our bioinformatic analysis indicates the En2-/- mouse is a valuable tool for investigating molecular alterations related to ASD.