Therapeutic irradiation of the brain is a common treatment modality for brain tumors, but can lead to impairment of cognitive function. Dendritic spines are sites of excitatory synaptic transmission and changes in spine structure and number are thought to represent a morphological correlate of altered brain functions associated with hippocampal dependent learning and memory. To gain some insight into the temporal and sub region specific cellular changes in the hippocampus following brain irradiation, we investigated the effects of 10 Gy cranial irradiation on dendritic spines in young adult mice. One week or 1 month post irradiation, changes in spine density and morphology in dentate gyrus (DG) granule and CA1 pyramidal neurons were quantified using Golgi staining. Our results showed that in the DG, there were significant reductions in spine density at both 1 week (11.9%) and 1 month (26.9%) after irradiation. In contrast, in the basal dendrites of CA1 pyramidal neurons, irradiation resulted in a significant reduction (18.7%) in spine density only at 1 week post irradiation. Analysis of spine morphology showed that irradiation led to significant decreases in the proportion of mushroom spines at both time points in the DG as well as CA1 basal dendrites. The proportions of stubby spines were significantly increased in both the areas at 1 month post irradiation. Irradiation did not alter spine density in the CA1 apical dendrites, but there were significant changes in the proportion of thin and mushroom spines at both time points post irradiation. Although the mechanisms involved are not clear, these findings are the first to show that brain irradiation of young adult animals leads to alterations in dendritic spine density and morphology in the hippocampus in a time dependent and region specific manner.

STEP (STriatal-Enriched protein tyrosine Phosphatase) is a neuron-specific phosphatase that regulates N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking, as well as ERK1/2, p38, Fyn, and Pyk2 activity. STEP is overactive in several neuropsychiatric and neurodegenerative disorders, including Alzheimer's disease (AD). The increase in STEP activity likely disrupts synaptic function and contributes to the cognitive deficits in AD. AD mice lacking STEP have restored levels of glutamate receptors on synaptosomal membranes and improved cognitive function, results that suggest STEP as a novel therapeutic target for AD. Here we describe the first large-scale effort to identify and characterize small-molecule STEP inhibitors. We identified the benzopentathiepin 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (known as TC-2153) as an inhibitor of STEP with an IC50 of 24.6 nM. TC-2153 represents a novel class of PTP inhibitors based upon a cyclic polysulfide pharmacophore that forms a reversible covalent bond with the catalytic cysteine in STEP. In cell-based secondary assays, TC-2153 increased tyrosine phosphorylation of STEP substrates ERK1/2, Pyk2, and GluN2B, and exhibited no toxicity in cortical cultures. Validation and specificity experiments performed in wild-type (WT) and STEP knockout (KO) cortical cells and in vivo in WT and STEP KO mice suggest specificity of inhibitors towards STEP compared to highly homologous tyrosine phosphatases. Furthermore, TC-2153 improved cognitive function in several cognitive tasks in 6- and 12-mo-old triple transgenic AD (3xTg-AD) mice, with no change in beta amyloid and phospho-tau levels.

Striatal-Enriched protein tyrosine Phosphatase of MW 61 kDa (STEP(61)) is a protein tyrosine phosphatase recently implicated in the pathophysiology of Alzheimer's disease (AD). STEP(61) is elevated in human AD prefrontal cortex and in the cortex of several AD mouse models. The elevated levels of active STEP(61) down-regulate surface expression of GluN1/GluN2B (formerly NR1/NR2B) receptor complexes, while genetically reducing STEP levels rescues both the biochemical and cognitive deficits in a triple transgenic AD mouse model (3xTg-AD). Here, we show that increased STEP(61) also plays a role in beta amyloid (Aβ)-mediated internalization of the α-amino-3-hydroxy-5-methyl-4-(AMPA) receptor (AMPAR) subunits GluA1/GluA2 (formerly GluR1/GluR2). We purified Aβ oligomers and determined that oligomers, but not monomers, lead to endocytosis of GluA1/GluA2 receptors in cortical cultures. The decrease in GluA1/GluA2 receptors is reversed in the progeny of STEP knock-out (KO) mice crossed with Tg2576 mice, despite elevated levels of Aβ. These results provide strong support for the hypothesis that STEP(61) is required for Aβ-mediated internalization of GluA1/GluA2 receptors.

Investigations into the causative role that western dietary patterns have on obesity and disease pathogenesis have speculated that quality and quantity of dietary fats and/or carbohydrates have a predictive role in the development of these disorders. Standard reference diets such as the AIN-93 rodent diet have historically been used to promote animal health and reduce variation of results across experiments, rather than model modern human dietary habits or nutrition-related pathologies. In rodents high-fat diets (HFDs) became a classic tool to investigate diet-induced obesity (DIO). These murine diets often relied on a single fat source with the most DIO consistent HFDs containing levels of fat up to 45-60% (kcal), higher than the reported human intake of 33-35% (kcal). More recently, researchers are formulating experimental animal (pre-clinical) diets that reflect mean human macro- and micronutrient consumption levels described by the National Health and Nutrition Examination Survey (NHANES). These diets attempt to integrate relevant ingredient sources and levels of nutrients; however, they most often fail to include high-fructose corn syrup (HFCS) as a source of dietary carbohydrate. We have formulated a modified Standard American Diet (mSAD) that incorporates relevant levels and sources of nutrient classes, including dietary HFCS, to assess the basal physiologies associated with mSAD consumption. Mice proffered the mSAD for 15 weeks displayed a phenotype consistent with metabolic syndrome, exhibiting increased adiposity, fasting hyperglycemia with impaired glucose and insulin tolerance. Metabolic alterations were evidenced at the tissue level as crown-like structures (CLS) in adipose tissue and fatty acid deposition in the liver, and targeted 16S rRNA metagenomics revealed microbial compositional shifts between dietary groups. This study suggests diet quality significantly affects metabolic homeostasis, emphasizing the importance of developing relevant pre-clinical diets to investigate chronic diseases highly impacted by western dietary consumption patterns.

Uncontrolled radiation exposure due to radiological terrorism, industrial accidents or military circumstances is a continuing threat for the civilian population. Age plays a major role in the susceptibility to radiation; younger children are at higher risk of developing cognitive deterioration when compared to adults. Our objective was to determine if an exposure to radiation affected the vulnerability of the juvenile hippocampus to a subsequent moderate traumatic injury.

Millions of traumatic brain injuries (TBIs) occur annually. TBIs commonly result from falls, traffic accidents, and sports-related injuries, all of which involve rotational acceleration/deceleration of the brain. During these injuries, the brain endures a multitude of primary insults including compression of brain tissue, damaged vasculature, and diffuse axonal injury. All of these deleterious effects can contribute to secondary brain ischemia, cellular death, and neuroinflammation that progress for weeks, months, and lifetime after injury. While the linear effects of head trauma have been extensively modeled, less is known about how rotational injuries mediate neuronal damage following injury. Here, we developed a new model of repetitive rotational head trauma in rodents and demonstrated acute and prolonged pathological, behavioral, and electrophysiological effects of rotational TBI (rTBI). We identify aberrant Cyclin-dependent kinase 5 (Cdk5) activity as a principal mediator of rTBI. We utilized Cdk5-enriched phosphoproteomics to uncover potential downstream mediators of rTBI and show pharmacological inhibition of Cdk5 reduces the cognitive and pathological consequences of injury. These studies contribute meaningfully to our understanding of the mechanisms of rTBI and how they may be effectively treated.

Cortical glutamate and midbrain dopamine neurotransmission converge to mediate striatum-dependent behaviors, while maladaptations in striatal circuitry contribute to mental disorders. However, the crosstalk between glutamate and dopamine signaling has not been entirely elucidated. Here we uncover a molecular mechanism by which glutamatergic and dopaminergic signaling integrate to regulate cAMP-dependent protein kinase (PKA) via phosphorylation of the PKA regulatory subunit, RIIβ. Using a combination of biochemical, pharmacological, neurophysiological, and behavioral approaches, we find that glutamate-dependent reduction in cyclin-dependent kinase 5 (Cdk5)-dependent RIIβ phosphorylation alters the PKA holoenzyme autoinhibitory state to increase PKA signaling in response to dopamine. Furthermore, we show that disruption of RIIβ phosphorylation by Cdk5 enhances cortico-ventral striatal synaptic plasticity. In addition, we demonstrate that acute and chronic stress in rats inversely modulate RIIβ phosphorylation and ventral striatal infusion of a small interfering peptide that selectively targets RIIβ regulation by Cdk5 improves behavioral response to stress. We propose this new signaling mechanism integrating ventral striatal glutamate and dopamine neurotransmission is important to brain function, may contribute to neuropsychiatric conditions, and serves as a possible target for the development of novel therapeutics for stress-related disorders.

Brain-derived neurotrophic factor (BDNF) regulates synaptic strengthening and memory consolidation, and altered BDNF expression is implicated in a number of neuropsychiatric and neurodegenerative disorders. BDNF potentiates N-methyl-D-aspartate receptor function through activation of Fyn and ERK1/2. STriatal-Enriched protein tyrosine Phosphatase (STEP) is also implicated in many of the same disorders as BDNF but, in contrast to BDNF, STEP opposes the development of synaptic strengthening. STEP-mediated dephosphorylation of the NMDA receptor subunit GluN2B promotes internalization of GluN2B-containing NMDA receptors, while dephosphorylation of the kinases Fyn, Pyk2, and ERK1/2 leads to their inactivation. Thus, STEP and BDNF have opposing functions. In this study, we demonstrate that manipulation of BDNF expression has a reciprocal effect on STEP61 levels. Reduced BDNF signaling leads to elevation of STEP61 both in BDNF(+/-) mice and after acute BDNF knockdown in cortical cultures. Moreover, a newly identified STEP inhibitor reverses the biochemical and motor abnormalities in BDNF(+/-) mice. In contrast, increased BDNF signaling upon treatment with a tropomyosin receptor kinase B agonist results in degradation of STEP61 and a subsequent increase in the tyrosine phosphorylation of STEP substrates in cultured neurons and in mouse frontal cortex. These findings indicate that BDNF-tropomyosin receptor kinase B signaling leads to degradation of STEP61 , while decreased BDNF expression results in increased STEP61 activity. A better understanding of the opposing interaction between STEP and BDNF in normal cognitive functions and in neuropsychiatric disorders will hopefully lead to better therapeutic strategies. Altered expression of BDNF and STEP61 has been implicated in several neurological disorders. BDNF and STEP61 are known to regulate synaptic strengthening, but in opposite directions. Here, we report that reduced BDNF signaling leads to elevation of STEP61 both in BDNF(+/-) mice and after acute BDNF knockdown in cortical cultures. In contrast, activation of TrkB receptor results in the degradation of STEP61 and reverses hyperlocomotor activity in BDNF(+/-) mice. Moreover, inhibition of STEP61 by TC-2153 is sufficient to enhance the Tyr phosphorylation of STEP substrates and also reverses hyperlocomotion in BDNF(+/-) mice. These findings give us a better understanding of the regulation of STEP61 by BDNF in normal cognitive functions and in neuropsychiatric disorders.

Cyclin-dependent kinase 5 (Cdk5) is a crucial regulator of neuronal signal transduction. Cdk5 activity is implicated in various neuropsychiatric and neurodegenerative conditions such as stress, anxiety, depression, addiction, Alzheimer's disease, and Parkinson's disease. While constitutive Cdk5 knockout is perinatally lethal, conditional knockout mice display resilience to stress-induction, enhanced cognition, neuroprotection from stroke and head trauma, and ameliorated neurodegeneration. Thus, Cdk5 represents a prime target for treatment in a spectrum of neurological and neuropsychiatric conditions. While intracranial infusions or treatment of acutely dissected brain tissue with compounds that inhibit Cdk5 have allowed the study of kinase function and corroborated conditional knockout findings, potent brain-penetrant systemically deliverable Cdk5 inhibitors are extremely limited, and no Cdk5 inhibitor has been approved to treat any neuropsychiatric or degenerative diseases to date. Here, we screened aminopyrazole-based analogs as potential Cdk5 inhibitors and identified a novel analog, 25-106, as a uniquely brain-penetrant anti-Cdk5 drug. We characterize the pharmacokinetic and dynamic responses of 25-106 in mice and functionally validate the effects of Cdk5 inhibition on open field and tail-suspension behaviors. Altogether, 25-106 represents a promising preclinical Cdk5 inhibitor that can be systemically administered with significant potential as a neurological/neuropsychiatric therapeutic.

The tyrosine kinase Fyn has two regulatory tyrosine residues that when phosphorylated either activate (Tyr(420)) or inhibit (Tyr(531)) Fyn activity. Within the central nervous system, two protein tyrosine phosphatases (PTPs) target these regulatory tyrosines in Fyn. PTPα dephosphorylates Tyr(531) and activates Fyn, while STEP (STriatal-Enriched protein tyrosine Phosphatase) dephosphorylates Tyr(420) and inactivates Fyn. Thus, PTPα and STEP have opposing functions in the regulation of Fyn; however, whether there is cross talk between these two PTPs remains unclear. Here, we used molecular techniques in primary neuronal cultures and in vivo to demonstrate that STEP negatively regulates PTPα by directly dephosphorylating PTPα at its regulatory Tyr(789). Dephosphorylation of Tyr(789) prevents the translocation of PTPα to synaptic membranes, blocking its ability to interact with and activate Fyn. Genetic or pharmacologic reduction in STEP61 activity increased the phosphorylation of PTPα at Tyr(789), as well as increased translocation of PTPα to synaptic membranes. Activation of PTPα and Fyn and trafficking of GluN2B to synaptic membranes are necessary for ethanol (EtOH) intake behaviors in rodents. We tested the functional significance of STEP61 in this signaling pathway by EtOH administration to primary cultures as well as in vivo, and demonstrated that the inactivation of STEP61 by EtOH leads to the activation of PTPα, its translocation to synaptic membranes, and the activation of Fyn. These findings indicate a novel mechanism by which STEP61 regulates PTPα and suggest that STEP and PTPα coordinate the regulation of Fyn. STEP61 , PTPα, Fyn, and NMDA receptor (NMDAR) have been implicated in ethanol intake behaviors in the dorsomedial striatum (DMS) in rodents. Here, we report that PTPα is a novel substrate for STEP61. Upon ethanol exposure, STEP61 is phosphorylated and inactivated by protein kinase A (PKA) signaling in the DMS. As a result of STEP61 inhibition, there is an increase in the phosphorylation of PTPα, which translocates to lipid rafts and activates Fyn and subsequent NMDAR signaling. The results demonstrate a synergistic regulation of Fyn-NMDAR signaling by STEP61 and PTPα, which may contribute to the regulation of ethanol-related behaviors. NMDA, N-methyl-D-aspartate; PTPα, receptor-type protein tyrosine phosphatase alpha; STEP, STriatal-Enriched protein tyrosine Phosphatase.

A unique feature of the space radiation environment is the presence of high-energy charged particles, including (56)Fe ions, which can present a significant hazard to space flight crews during and following a mission. (56)Fe irradiation-induced cognitive changes often involve alterations in hippocampal function. These alterations might involve changes in spine morphology and density. In addition to irradiation, performing a cognitive task can also affect spine morphology. Therefore, it is often hard to determine whether changes in spine morphology and density are due to an environmental challenge or group differences in performance on cognitive tests. In this study, we tested the hypothesis that the ability of exploratory behavior to increase specific measures of hippocampal spine morphology and density is affected by (56)Fe irradiation. In sham-irradiated mice, exploratory behavior increased basal spine density in the CA1 region of the hippocampus and the enclosed blade of the dentate gyrus. These effects were not seen in irradiated mice. In addition, following exploratory behavior, there was a trend toward a decrease in the percent stubby spines on apical dendrites in the CA3 region of the hippocampus in (56)Fe-irradiated, but not sham-irradiated, mice. Other hippocampal regions and spine measures affected by (56)Fe irradiation showed comparable radiation effects in behaviorally naïve and cognitively tested mice. Thus, the ability of exploratory behavior to alter spine density and morphology in specific hippocampal regions is affected by (56)Fe irradiation.

The neurobiological mechanisms that mediate psychiatric comorbidities associated with metabolic disorders such as obesity, metabolic syndrome and diabetes remain obscure. High fructose corn syrup (HFCS) is widely used in beverages and is often included in food products with moderate or high fat content that have been linked to many serious health issues including diabetes and obesity. However, the impact of such foods on the brain has not been fully characterized. Here, we evaluated the effects of long-term consumption of a HFCS-Moderate Fat diet (HFCS-MFD) on behavior, neuronal signal transduction, gut microbiota, and serum metabolomic profile in mice to better understand how its consumption and resulting obesity and metabolic alterations relate to behavioral dysfunction. Mice fed HFCS-MFD for 16 weeks displayed enhanced anxiogenesis, increased behavioral despair, and impaired social interactions. Furthermore, the HFCS-MFD induced gut microbiota dysbiosis and lowered serum levels of serotonin and its tryptophan-based precursors. Importantly, the HFCS-MFD altered neuronal signaling in the ventral striatum including reduced inhibitory phosphorylation of glycogen synthase kinase 3β (GSK3β), increased expression of ΔFosB, increased Cdk5-dependent phosphorylation of DARPP-32, and reduced PKA-dependent phosphorylation of the GluR1 subunit of the AMPA receptor. These findings suggest that HFCS-MFD-induced changes in the gut microbiota and neuroactive metabolites may contribute to maladaptive alterations in ventral striatal function that underlie neurobehavioral impairment. While future studies are essential to further evaluate the interplay between these factors in obesity and metabolic syndrome-associated behavioral comorbidities, these data underscore the important role of peripheral-CNS interactions in diet-induced behavioral and brain function. This study also highlights the clinical need to address neurobehavioral comorbidities associated with obesity and metabolic syndrome.

Mechanistic modeling of cancers such as Medullary Thyroid Carcinoma (MTC) to emulate patient-specific phenotypes is challenging. The discovery of potential diagnostic markers and druggable targets in MTC urgently requires clinically relevant animal models. Here we established orthotopic mouse models of MTC driven by aberrantly active Cdk5 using cell-specific promoters. Each of the two models elicits distinct growth differences that recapitulate the less or more aggressive forms of human tumors. The comparative mutational and transcriptomic landscape of tumors revealed significant alterations in mitotic cell cycle processes coupled with the slow-growing tumor phenotype. Conversely, perturbation in metabolic pathways emerged as critical for aggressive tumor growth. Moreover, an overlapping mutational profile was identified between mouse and human tumors. Gene prioritization revealed putative downstream effectors of Cdk5 which may contribute to the slow and aggressive growth in the mouse MTC models. In addition, Cdk5/p25 phosphorylation sites identified as biomarkers for Cdk5-driven neuroendocrine tumors (NETs) were detected in both slow and rapid onset models and were also histologically present in human MTC. Thus, this study directly relates mouse and human MTC models and uncovers vulnerable pathways potentially responsible for differential tumor growth rates. Functional validation of our findings may lead to better prediction of patient-specific personalized combinational therapies.