VTA multifaceted modulation of CA1 local circuits.

Excitatory pyramidal (PYR) cell activation of interneurons (INT) produces network oscillations that underlie cognitive processes in the hippocampus (CA1). Neural projections from the ventral tegmental area (VTA) to the hippocampus contribute to novelty detection by modulating CA1 PYR and INT activity. The role of the VTA in the VTA-hippocampus loop is mostly attributed to the dopamine neurons although the VTA glutamate-releasing terminals are dominant in the hippocampus. Because of the traditional focus on VTA dopamine circuits, how VTA glutamate inputs modulate PYR activation of INT in CA1 neuronal ensembles is poorly understood and has not been distinguished from the VTA dopamine inputs. By combining CA1 extracellular recording with VTA photostimulation in anesthetized mice, we compared the effects of VTA dopamine and glutamate input on CA1 PYR/INT connections. Stimulation of VTA glutamate neurons shortened PYR/INT connection time without altering the synchronization or connectivity strength. Conversely, activation of VTA dopamine inputs delayed CA1 PYR/INT connection time and increased the synchronization in putative pairs. Taken together, we conclude that VTA dopamine and glutamate projections produce tract-specific effects on CA1 PYR/INT connectivity and synchrony. As such, selective activation or co-activation of these systems will likely produce a range of modulatory effects on local CA1 circuits.

Homologous Recombination Deficiency (HRD) in Cutaneous Oncology.

Skin cancers, including basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (SCC), and melanoma, are the most common malignancies in the United States. Loss of DNA repair pathways in the skin plays a significant role in tumorigenesis. In recent years, targeting DNA repair pathways, particularly homologous recombination deficiency (HRD), has emerged as a potential therapeutic approach in cutaneous malignancies. This review provides an overview of DNA damage and repair pathways, with a focus on HRD, and discusses major advances in targeting these pathways in skin cancers. Poly(ADP-ribose) polymerase (PARP) inhibitors have been developed to exploit HRD in cancer cells. PARP inhibitors disrupt DNA repair mechanisms by inhibiting PARP enzymatic activity, leading to the accumulation of DNA damage and cell death. The concept of synthetic lethality has been demonstrated in HR-deficient cells, such as those with BRCA1/2 mutations, which exhibit increased sensitivity to PARP inhibitors. HRD assessment methods, including genomic scars, RAD51 foci formation, functional assays, and BRCA1/2 mutation analysis, are discussed as tools for identifying patients who may benefit from PARP inhibitor therapy. Furthermore, HRD has been implicated in the response to immunotherapy, and the combination of PARP inhibitors with immunotherapy has shown promising results. The frequency of HRD in melanoma ranges from 18% to 57%, and studies investigating the use of PARP inhibitors as monotherapy in melanoma are limited. Further research is warranted to explore the potential of PARP inhibition in melanoma treatment.

VTA Excitatory Neurons Control Reward-driven Behavior by Modulating Infralimbic Cortical Firing.

The functional dichotomy of anatomical regions of the medial prefrontal cortex (mPFC) has been tested with greater certainty in punishment-driven tasks, and less so in reward-oriented paradigms. In the infralimbic cortex (IL), known for behavioral suppression (STOP), tasks linked with reward or punishment are encoded through firing rate decrease or increase, respectively. Although the ventral tegmental area (VTA) is the brain region governing reward/aversion learning, the link between its excitatory neuron population and IL encoding of reward-linked behavioral expression is unclear. Here, we present evidence that IL ensembles use a population-based mechanism involving broad inhibition of principal cells at intervals when reward is presented or expected. The IL encoding mechanism was consistent across multiple sessions with randomized rewarded target sites. Most IL neurons exhibit FR (Firing Rate) suppression during reward acquisition intervals (T1), and subsequent exploration of previously rewarded targets when the reward is omitted (T2). Furthermore, FR suppression in putative IL ensembles persisted for intervals that followed reward-linked target events. Pairing VTA glutamate inhibition with reward acquisition events reduced the weight of reward-target association expressed as a lower affinity for previously rewarded targets. For these intervals, fewer IL neurons per mouse trial showed FR decrease and were accompanied by an increase in the percentage of units with no change in FR. Together, we conclude that VTA glutamate neurons are likely involved in establishing IL inhibition states that encode reward acquisition, and subsequent reward-target association.

Prenatal and postnatal methamphetamine exposure alters prefrontal cortical gene expression and behavior in mice.

Methamphetamine is a highly abused psychostimulant that substantially impacts public health. Prenatal and postnatal methamphetamine exposure alters gene expression, brain development, and behavior in the offspring, although the underlying mechanisms are not fully defined. To assess these adverse outcomes in the offspring, we employed a mouse model of prenatal and postnatal methamphetamine exposure. Juvenile offspring were behaviorally assessed on the open field, novel object recognition, Y-maze, and forced swim tests. In addition, RNA sequencing was used to explore potential alterations in prefrontal cortical gene expression. We found that methamphetamine-exposed mice exhibited decreased locomotor activity and impaired cognitive performance. In addition, differential expression of genes involved in neurotransmission, synaptic plasticity, and neuroinflammation were found with notable changes in dopaminergic signaling pathways. These data suggest potential neural and molecular mechanisms underlying methamphetamine-exposed behavioral changes. The altered expression of genes involved in dopaminergic signaling and synaptic plasticity highlights potential targets for therapeutic interventions for substance abuse disorders and related psychiatric complications.

Genomic and Transcriptomic Landscape of RET Wild-Type Medullary Thyroid Cancer and Potential Use of Mitogen-Activated Protein Kinase-Targeted Therapy.

BACKGROUND: About 75% of medullary thyroid cancers (MTCs) are sporadic with 45% to 70% being driven by a RET mutation. Selpercatinib is an approved treatment for RET-mutated (mut RET ) MTC; however, treatments are needed for wild-type RET MTC (wt RET ). Genomic alterations and transcriptomic signatures of wt RET MTC may reveal new therapeutic insights. STUDY DESIGN: We did a retrospective analysis of MTC samples submitted for DNA/RNA sequencing and programmed cell death ligand 1 expression using immunohistochemistry at a Clinical Laboratory Improvement Amendments/College of American Pathologists-certified laboratory. Tumor microenvironment immune cell fractions were estimated using RNA deconvolution (quanTIseq). Transcriptomic signatures of inflammation and MAP kinase pathway activation scores were calculated. Mann-Whitney U, chi-square, and Fisher's exact tests were applied (p values adjusted for multiple comparisons). RESULTS: The 160-patient cohort included 108 mut RET and 52 wt RET MTC samples. wt RET tumors frequently harbored mitogen-activated protein kinase (MAPK) pathway mutations, including HRAS (42.31%), KRAS (15.7%), NF1 (6.7%), and BRAF (2%), whereas only 1 MAPK pathway mutation ( NF1 ) was identified among mut RET MTC. Recurrent mutations seen in wt RET MTC included MGA , VHL, APC , STK11 , and NFE2L2 . Increased transcriptional activation of the MAPK pathway was observed in patients with wt RET harboring mutations in MAPK genes. Although the frequency of programmed cell death ligand 1 expression was similar in wt RET and mut RET (10.2% vs 7%, p = 0.531), wt RET tumors were more often tumor mutational burden high (7.7% vs 0%, p = 0.011), and wt RET MTC exhibited higher expression of immune checkpoint genes. CONCLUSIONS: We identified molecular alterations and immune-related features that distinguish wt RET from mut RET MTC. Although RET mutation drives MTC in the absence of other alterations, we showed that wt RET MTC frequently harbors MAPK pathway mutations. These findings may indicate a potential basis for MAPK-targeted therapy, possibly in combination with immuno-oncology agents for selected patients with wt RET MTC.

ARID2 mutations may relay a distinct subset of cutaneous melanoma patients with different outcomes.

ARID genes encode subunits of SWI/SNF chromatin remodeling complexes and are frequently mutated in human cancers. We investigated the correlation between ARID mutations, molecular features, and clinical outcomes in melanoma patients. Cutaneous melanoma samples (n = 1577) were analyzed by next-generation sequencing. Samples were stratified by pathogenic/likely pathogenic mutation in ARID genes (ARID1A/2/1B/5B). PD-L1 expression was assessed using IHC (SP142; positive (+): ≥ 1%). Tumor mutation burden (TMB)-high was defined as ≥ 10 mutations/Mb. Transcriptomic signatures predictive of response to immune checkpoint inhibitors-interferon gamma and T-cell inflamed score were calculated. Real-world overall survival (OS) information was obtained from insurance claims data, with Kaplan-Meier estimates calculated from time of tissue collection until last date of contact. Mann-Whitney U, Chi-square, and Fisher exact tests were applied where appropriate, with p values adjusted for multiple comparisons. ARID2 mutations were more prevalent in cutaneous melanoma compared to ARID1A (11.0%: n = 451 vs 2.8%: n = 113), with concurrent ARID1A/ARID2 mutation in 1.1% (n = 46) of samples. ARID mutations were associated with a high prevalence of RAS pathway mutations-NF1 (ARID1A, 52.6%; ARID2, 48.5%; ARID1A/2, 63.6%; and ARID-WT, 13.3%; p < 0.0001) and KRAS (ARID1A, 3.5%; ARID2, 3.1%; ARID1A/2, 6.5%; and ARID-WT, 1.0%; p = 0.018)), although BRAF mutations were less common in ARID-mutated cohorts (ARID1A, 31.9%; ARID2, 35.6%; ARID1A/2, 26.1%; and ARID-WT, 50.4%; p < 0.0001). TMB-high was more common in ARID-mutated samples (ARID1A, 80.9%; ARID2, 89.9%; ARID1A/2, 100%; and ARID-WT, 49.4%; p < 0.0001), while PD-L1 positivity was similar across subgroups (ARID1A, 43.8%; ARID2, 51.1%; ARID1A/2, 52.5%; and ARID-WT, 44.9%; p = 0.109). Patients with ARID1A mutations had a higher prevalence of dMMR/MSI-H compared to those with ARID-WT (2.7% vs 0.2%, p = 0.030). Median IFN-γ and T-cell signatures were higher in ARID2-mutated samples compared to ARID-WT (IFN-γ: - 0.15 vs - 0.21, p = 0.0066; T-cell: 23.5 vs - 18.5, p = 0.041). ARID2-mutated patients had improved survival compared to ARID-WT; (HR: 1.22 (95% CI 1.0-1.5), p = 0.022). No additional OS benefit was observed with anti-PD-1 therapy for ARID2 mutation compared to ARID-WT. Melanoma patients with ARID mutations exhibited higher prevalence of markers associated with ICI response, including TMB-H, and immune-related signatures. Our data also suggests improved survival outcome in patients with ARID2 mutations, irrespective of anti-PD1 therapy.

Neuroanatomical Alterations in the CNTNAP2 Mouse Model of Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is associated with neurodevelopmental alterations, including atypical forebrain cellular organization. Mutations in several ASD-related genes often result in cerebral cortical anomalies, such as the abnormal developmental migration of excitatory pyramidal cells and the malformation of inhibitory neuronal circuitry. Notably here, mutations in the CNTNAP2 gene result in ectopic superficial cortical neurons stalled in lower cortical layers and alterations to the balance of cortical excitation and inhibition. However, the broader circuit-level implications of these findings have not been previously investigated. Therefore, we assessed whether ectopic cortical neurons in CNTNAP2 mutant mice form aberrant connections with higher-order thalamic nuclei, potentially accounting for some autistic behaviors, such as repetitive and hyperactive behaviors. Furthermore, we assessed whether the development of parvalbumin-positive (PV) cortical interneurons and their specialized matrix support structures, called perineuronal nets (PNNs), were altered in these mutant mice. We found alterations in both ectopic neuronal connectivity and in the development of PNNs, PV neurons and PNNs enwrapping PV neurons in various sensory cortical regions and at different postnatal ages in the CNTNAP2 mutant mice, which likely lead to some of the cortical excitation/inhibition (E/I) imbalance associated with ASD. These findings suggest neuroanatomical alterations in cortical regions that underlie the emergence of ASD-related behaviors in this mouse model of the disorder.

Behavioral regulation by perineuronal nets in the prefrontal cortex of the CNTNAP2 mouse model of autism spectrum disorder.

Autism spectrum disorders (ASDs) arise from altered development of the central nervous system, and manifest behaviorally as social interaction deficits and restricted and repetitive behaviors. Alterations to parvalbumin (PV) expressing interneurons have been implicated in the neuropathological and behavioral deficits in autism. In addition, perineuronal nets (PNNs), specialized extracellular matrix structures that enwrap the PV-expressing neurons, also may be altered, which compromises neuronal function and susceptibility to oxidative stress. In particular, the prefrontal cortex (PFC), which regulates several core autistic traits, relies on the normal organization of PNNs and PV-expressing cells, as well as other neural circuit elements. Consequently, we investigated whether PNNs and PV-expressing cells were altered in the PFC of the CNTNAP2 knockout mouse model of ASD and whether these contributed to core autistic-like behaviors in this model system. We observed an overexpression of PNNs, PV-expressing cells, and PNNs enwrapping PV-expressing cells in adult CNTNAP2 mice. Transient digestion of PNNs from the prefrontal cortex (PFC) by injection of chondroitinase ABC in CNTNAP2 mutant mice rescued some of the social interaction deficits, but not the restricted and repetitive behaviors. These findings suggest that the neurobiological regulation of PNNs and PVs in the PFC contribute to social interaction behaviors in neurological disorders including autism.

KinFams: De-Novo Classification of Protein Kinases Using CATH Functional Units.

Protein kinases are important targets for treating human disorders, and they are the second most targeted families after G-protein coupled receptors. Several resources provide classification of kinases into evolutionary families (based on sequence homology); however, very few systematically classify functional families (FunFams) comprising evolutionary relatives that share similar functional properties. We have developed the FunFam-MARC (Multidomain ARchitecture-based Clustering) protocol, which uses multi-domain architectures of protein kinases and specificity-determining residues for functional family classification. FunFam-MARC predicts 2210 kinase functional families (KinFams), which have increased functional coherence, in terms of EC annotations, compared to the widely used KinBase classification. Our protocol provides a comprehensive classification for kinase sequences from >10,000 organisms. We associate human KinFams with diseases and drugs and identify 28 druggable human KinFams, i.e., enriched in clinically approved drugs. Since relatives in the same druggable KinFam tend to be structurally conserved, including the drug-binding site, these KinFams may be valuable for shortlisting therapeutic targets. Information on the human KinFams and associated 3D structures from AlphaFold2 are provided via our CATH FTP website and Zenodo. This gives the domain structure representative of each KinFam together with information on any drug compounds available. For 32% of the KinFams, we provide information on highly conserved residue sites that may be associated with specificity.

Anterior thalamic glutamatergic neurons modulate prefrontal cortical firing and regularity.

For decades, studies on learning and memory focused extensively on the neural circuitry between the hippocampus and the prefrontal cortex, with minimal consideration of the role of the anterior thalamic nucleus (ATN). The diencephalic nucleus is rich in excitatory glutamate neurons that project to both the hippocampus and medial prefrontal cortex (mPFC). Neural projections from the hippocampus and mPFC have also been identified in the ATN through reciprocal circuits. Although ATN lesioning leads to memory loss (amnesia), the role of the ATN in the propagation of cognitive processes in the mPFC is still poorly understood. In the current study, we employed adeno-associated viral labeling and in vivo electrophysiology to trace ATN glutamate neural projections in the layers of the mPFC. Neuroanatomical mapping of ATN Vglut2+ projections revealed a topographic gradient in the infralimbic cortex (IL), prelimbic cortex (PrL), and cingulate cortex (Cg1). Specifically, the IL has the least ATN Vglut2+ terminals while the PrL (layer III) and Cg1 (layers III, Va/b, VI) have robust innervation. Functional tracing of the thalamocortical projection was performed by combining extracellular Cg1 and ATN recording with ATN Vglut2+ neuron photostimulation. Our results show that light-activated ATN Vglut2+ neurons drive Cg1 neural activity and increase the firing rate of putative pyramidal neurons in anesthetized mice. In addition, Cg1 putative neurons that were activated during ATN photostimulation show a significant increase in firing regularity in comparison with the baseline (no ATN stimulus). Together, our results show that ATN Vglut2+ neurons modulate the firing rate and regularity of the putative Cg1 pyramidal cells, thus, creating a premise for direct influence on cognitive processes in cortical networks.

Exploiting protein family and protein network data to identify novel drug targets for bladder cancer.

Bladder cancer remains one of the most common forms of cancer and yet there are limited small molecule targeted therapies. Here, we present a computational platform to identify new potential targets for bladder cancer therapy. Our method initially exploited a set of known driver genes for bladder cancer combined with predicted bladder cancer genes from mutationally enriched protein domain families. We enriched this initial set of genes using protein network data to identify a comprehensive set of 323 putative bladder cancer targets. Pathway and cancer hallmarks analyses highlighted putative mechanisms in agreement with those previously reported for this cancer and revealed protein network modules highly enriched in potential drivers likely to be good targets for targeted therapies. 21 of our potential drug targets are targeted by FDA approved drugs for other diseases - some of them are known drivers or are already being targeted for bladder cancer (FGFR3, ERBB3, HDAC3, EGFR). A further 4 potential drug targets were identified by inheriting drug mappings across our in-house CATH domain functional families (FunFams). Our FunFam data also allowed us to identify drug targets in families that are less prone to side effects i.e., where structurally similar protein domain relatives are less dispersed across the human protein network. We provide information on our novel potential cancer driver genes, together with information on pathways, network modules and hallmarks associated with the predicted and known bladder cancer drivers and we highlight those drivers we predict to be likely drug targets.

CA1 Spike Timing is Impaired in the 129S Inbred Strain During Cognitive Tasks.

A spontaneous mutation of the disrupted in schizophrenia 1 (Disc1) gene is carried by the 129S inbred mouse strain. Truncated DISC1 protein in 129S mouse synapses impairs the scaffolding of excitatory postsynaptic receptors and leads to progressive spine dysgenesis. In contrast, C57BL/6 inbred mice carry the wild-type Disc1 gene and exhibit more typical cognitive performance in spatial exploration and executive behavioral tests. Because of the innate Disc1 mutation, adult 129S inbred mice exhibit the behavioral phenotypes of outbred B6 Disc1 knockdown (Disc1-/-) or Disc1-L-100P mutant strains. Recent studies in Disc1-/- and L-100P mice have shown that impaired excitation-driven interneuron activity and low hippocampal theta power underlie the behavioral phenotypes that resemble human depression and schizophrenia. The current study compared the firing rate and connectivity profile of putative neurons in the CA1 of freely behaving inbred 129S and B6 mice, which have mutant and wild-type Disc1 genes, respectively. In cognitive behavioral tests, 129S mice had lower exploration scores than B6 mice. Furthermore, the mean firing rate for 129S putative pyramidal (pyr) cells and interneurons (int) was significantly lower than that for B6 CA1 neurons sampled during similar tasks. Analysis of pyr/int connectivity revealed a significant delay in synaptic transmission for 129S putative pairs. Sampled 129S pyr/int pairs also had lower detectability index scores than B6 putative pairs. Therefore, the spontaneous Disc1 mutation in the 129S strain attenuates the firing of putative pyr CA1 neurons and impairs spike timing fidelity during cognitive tasks.

Crossed Connections From Insular Cortex to the Contralateral Thalamus.

Sensory information in all modalities, except olfaction, is processed at the level of the thalamus before subsequent transmission to the cerebral cortex. This incoming sensory stream is refined and modulated in the thalamus by numerous descending corticothalamic projections originating in layer 6 that ultimately alter the sensitivity and selectivity for sensory features. In general, these sensory thalamo-cortico-thalamic loops are considered strictly unilateral, i.e., no contralateral crosstalk between cortex and thalamus. However, in contrast to this canonical view, we characterize here a prominent contralateral corticothalamic projection originating in the insular cortex, utilizing both retrograde tracing and cre-lox mediated viral anterograde tracing strategies with the Ntsr1-Cre transgenic mouse line. From our studies, we find that the insular contralateral corticothalamic projection originates from a separate population of layer 6 neurons than the ipsilateral corticothalamic projection. Furthermore, the contralateral projection targets a topographically distinct subregion of the thalamus than the ipsilateral projection. These findings suggest a unique bilateral mechanism for the top-down refinement of ascending sensory information.

A CATH domain functional family based approach to identify putative cancer driver genes and driver mutations.

Tumour sequencing identifies highly recurrent point mutations in cancer driver genes, but rare functional mutations are hard to distinguish from large numbers of passengers. We developed a novel computational platform applying a multi-modal approach to filter out passengers and more robustly identify putative driver genes. The primary filter identifies enrichment of cancer mutations in CATH functional families (CATH-FunFams) - structurally and functionally coherent sets of evolutionary related domains. Using structural representatives from CATH-FunFams, we subsequently seek enrichment of mutations in 3D and show that these mutation clusters have a very significant tendency to lie close to known functional sites or conserved sites predicted using CATH-FunFams. Our third filter identifies enrichment of putative driver genes in functionally coherent protein network modules confirmed by literature analysis to be cancer associated. Our approach is complementary to other domain enrichment approaches exploiting Pfam families, but benefits from more functionally coherent groupings of domains. Using a set of mutations from 22 cancers we detect 151 putative cancer drivers, of which 79 are not listed in cancer resources and include recently validated cancer associated genes EPHA7, DCC netrin-1 receptor and zinc-finger protein ZNF479.

Structural and Functional View of Polypharmacology.

Protein domains mediate drug-protein interactions and this principle can guide the design of multi-target drugs i.e. polypharmacology. In this study, we associate multi-target drugs with CATH functional families through the overrepresentation of targets of those drugs in CATH functional families. Thus, we identify CATH functional families that are currently enriched in drugs (druggable CATH functional families) and we use the network properties of these druggable protein families to analyse their association with drug side effects. Analysis of selected druggable CATH functional families, enriched in drug targets, show that relatives exhibit highly conserved drug binding sites. Furthermore, relatives within druggable CATH functional families occupy central positions in a human protein functional network, cluster together forming network neighbourhoods and are less likely to be within proteins associated with drug side effects. Our results demonstrate that CATH functional families can be used to identify drug-target interactions, opening a new research direction in target identification.