Case Report: Exceptional response to nivolumab plus cabozantinib in a patient with extrarenal clear cell renal cell carcinoma.

Extrarenal clear cell renal cell carcinoma (eccRCC) is a rare type of RCC that arises in areas other than the kidney. Given its rarity, consensus guidelines for optimal treatment of eccRCC have not been established, and the literature is lacking any reports of patient response to systemic therapy and any reports of administration of immunotherapy to patients with ecRCC. Here, we present the case of a patient in their 60s with eccRCC arising in the spleen. The patient underwent splenic resection and then received systemic therapy, due to disease recurrence, with a combination of immunotherapy (IO) and tyrosine kinase inhibitor targeted therapy (VEGF-TKI). The patient had an excellent and durable response to this therapeutic regimen with minimal adverse effects, completing 2 years of therapy of nivolumab and cabozantinib. At the time of this report, the disease remains stable. This case demonstrates that combination therapy with IO+VEGF-TKI represents a reasonable and well-tolerated treatment option with activity in eccRCC and reveals interesting correlative data, including nests of stem-like CD8+T-cell infiltration in tumor tissue, which provide important biological context to this patient's exceptional therapeutic response.

Drosophila melanogaster as a model to study age and sex differences in brain injury and neurodegeneration after mild head trauma.

Repetitive physical insults to the head, including those that elicit mild traumatic brain injury (mTBI), are a known risk factor for a variety of neurodegenerative conditions including Alzheimer's disease (AD), Parkinson's disease (PD), and chronic traumatic encephalopathy (CTE). Although most individuals who sustain mTBI typically achieve a seemingly full recovery within a few weeks, a subset experience delayed-onset symptoms later in life. As most mTBI research has focused on the acute phase of injury, there is an incomplete understanding of mechanisms related to the late-life emergence of neurodegeneration after early exposure to mild head trauma. The recent adoption of Drosophila-based brain injury models provides several unique advantages over existing preclinical animal models, including a tractable framework amenable to high-throughput assays and short relative lifespan conducive to lifelong mechanistic investigation. The use of flies also provides an opportunity to investigate important risk factors associated with neurodegenerative conditions, specifically age and sex. In this review, we survey current literature that examines age and sex as contributing factors to head trauma-mediated neurodegeneration in humans and preclinical models, including mammalian and Drosophila models. We discuss similarities and disparities between human and fly in aging, sex differences, and pathophysiology. Finally, we highlight Drosophila as an effective tool for investigating mechanisms underlying head trauma-induced neurodegeneration and for identifying therapeutic targets for treatment and recovery.

The disease-associated proteins Drosophila Nab2 and Ataxin-2 interact with shared RNAs and coregulate neuronal morphology.

Nab2 encodes the Drosophila melanogaster member of a conserved family of zinc finger polyadenosine RNA-binding proteins (RBPs) linked to multiple steps in post-transcriptional regulation. Mutation of the Nab2 human ortholog ZC3H14 gives rise to an autosomal recessive intellectual disability but understanding of Nab2/ZC3H14 function in metazoan nervous systems is limited, in part because no comprehensive identification of metazoan Nab2/ZC3H14-associated RNA transcripts has yet been conducted. Moreover, many Nab2/ZC3H14 functional protein partnerships remain unidentified. Here, we present evidence that Nab2 genetically interacts with Ataxin-2 (Atx2), which encodes a neuronal translational regulator, and that these factors coordinately regulate neuronal morphology, circadian behavior, and adult viability. We then present the first high-throughput identifications of Nab2- and Atx2-associated RNAs in Drosophila brain neurons using RNA immunoprecipitation-sequencing (RIP-Seq). Critically, the RNA interactomes of each RBP overlap, and Nab2 exhibits high specificity in its RNA associations in neurons in vivo, associating with a small fraction of all polyadenylated RNAs. The identities of shared associated transcripts (e.g., drk, me31B, stai) and of transcripts specific to Nab2 or Atx2 (e.g., Arpc2 and tea) promise insight into neuronal functions of, and genetic interactions between, each RBP. Consistent with prior biochemical studies, Nab2-associated neuronal RNAs are overrepresented for internal A-rich motifs, suggesting these sequences may partially mediate Nab2 target selection. These data support a model where Nab2 functionally opposes Atx2 in neurons, demonstrate Nab2 shares associated neuronal RNAs with Atx2, and reveal Drosophila Nab2 associates with a more specific subset of polyadenylated mRNAs than its polyadenosine affinity alone may suggest.

A protocol to detect neurodegeneration in Drosophila melanogaster whole-brain mounts using advanced microscopy.

Drosophila melanogaster is an excellent model organism to study neurodegeneration. Assessing evident neurodegeneration within the fly brain involves the laborious preparation of thin-sectioned H&E-stained heads to visualize brain vacuole degeneration. Here, we present an advanced microscopy-based protocol, without the need for sectioning, to detect vacuole degeneration within whole fly brains by applying commonly used stains to reveal the brain parenchyma. This approach preserves the whole-brain architecture and enables rapid, reproducible, and quantitative analyses of vacuole-like degeneration associated with specific brain regions. For complete details on the use and execution of this protocol, please refer to Behnke et al. (2021).

Repetitive mild head trauma induces activity mediated lifelong brain deficits in a novel Drosophila model.

Mild head trauma, including concussion, can lead to chronic brain dysfunction and degeneration but the underlying mechanisms remain poorly understood. Here, we developed a novel head impact system to investigate the long-term effects of mild head trauma on brain structure and function, as well as the underlying mechanisms in Drosophila melanogaster. We find that Drosophila subjected to repetitive head impacts develop long-term deficits, including impaired startle-induced climbing, progressive brain degeneration, and shortened lifespan, all of which are substantially exacerbated in female flies. Interestingly, head impacts elicit an elevation in neuronal activity and its acute suppression abrogates the detrimental effects in female flies. Together, our findings validate Drosophila as a suitable model system for investigating the long-term effects of mild head trauma, suggest an increased vulnerability to brain injury in female flies, and indicate that early altered neuronal excitability may be a key mechanism linking mild brain trauma to chronic degeneration.

Comparative Ultrastructural Analysis of Thalamocortical Innervation of the Primary Motor Cortex and Supplementary Motor Area in Control and MPTP-Treated Parkinsonian Monkeys.

The synaptic organization of thalamic inputs to motor cortices remains poorly understood in primates. Thus, we compared the regional and synaptic connections of vGluT2-positive thalamocortical glutamatergic terminals in the supplementary motor area (SMA) and the primary motor cortex (M1) between control and MPTP-treated parkinsonian monkeys. In controls, vGluT2-containing fibers and terminal-like profiles invaded layer II-III and Vb of M1 and SMA. A significant reduction of vGluT2 labeling was found in layer Vb, but not in layer II-III, of parkinsonian animals, suggesting a potential thalamic denervation of deep cortical layers in parkinsonism. There was a significant difference in the pattern of synaptic connectivity in layers II-III, but not in layer Vb, between M1 and SMA of control monkeys. However, this difference was abolished in parkinsonian animals. No major difference was found in the proportion of perforated versus macular post-synaptic densities at thalamocortical synapses between control and parkinsonian monkeys in both cortical regions, except for a slight increase in the prevalence of perforated axo-dendritic synapses in the SMA of parkinsonian monkeys. Our findings suggest that disruption of the thalamic innervation of M1 and SMA may underlie pathophysiological changes of the motor thalamocortical loop in the state of parkinsonism.

Neuropeptide VGF Promotes Maturation of Hippocampal Dendrites That Is Reduced by Single Nucleotide Polymorphisms.

The neuropeptide VGF (non-acronymic) is induced by brain-derived neurotrophic factor and promotes hippocampal neurogenesis, as well as synaptic activity. However, morphological changes induced by VGF have not been elucidated. Developing hippocampal neurons were exposed to VGF through bath application or virus-mediated expression in vitro. VGF-derived peptide, TLQP-62, enhanced dendritic branching, and outgrowth. Furthermore, VGF increased dendritic spine density and the proportion of immature spines. Spine formation was associated with increased synaptic protein expression and co-localization of pre- and postsynaptic markers. Three non-synonymous single nucleotide polymorphisms (SNPs) were selected in human VGF gene. Transfection of N2a cells with plasmids containing these SNPs revealed no relative change in protein expression levels and normal protein size, except for a truncated protein from the premature stop codon, E525X. All three SNPs resulted in a lower proportion of N2a cells bearing neurites relative to wild-type VGF. Furthermore, all three mutations reduced the total length of dendrites in developing hippocampal neurons. Taken together, our results suggest VGF enhances dendritic maturation and that these effects can be altered by common mutations in the VGF gene. The findings may have implications for people suffering from psychiatric disease or other conditions who may have altered VGF levels.

Autoantibodies in Human Diabetic Depression Inhibit Adult Neural Progenitor Cells In vitro and Induce Depressive-Like Behavior in Rodents.

AIM: Diabetic depression increases in association with microvascular complications. We tested a hypothesis that circulating autoantibodies having anti-endothelial and anti-neuronal properties increase in subsets of diabetes with co-morbid depression. METHODS: Protein-A eluates from plasma of 20 diabetic depression patients and 30 age-matched controls were tested for effects on endothelial cell survival, neurite outgrowth in rat pheochromocytoma (PC12) cells, or process extension and survival in adult rat dentate gyrus neural progenitor cells. The protein-A eluates from depressed or non-depressed, diabetic patients were injected (via intracerebroventricular route) into mice and 7-10 days later behavioral tests (sucrose preference, and tail suspension tests) were conducted to determine whether the autoantibodies induced anhedonia or despair. RESULTS: Diabetic depression (n=20) autoantibodies caused a significant inhibition of PC12 cell neurite outgrowth (P<0.001) or endothelial cell proliferation compared to autoantibodies in control, diabetic (n=20) or non-diabetic (n=10) patients without depression. Process extension and survival in adult rat dentate gyrus neural progenitor cells was significantly reduced (P<0.001) by diabetic depression autoantibodies (n= 11) compared to the effects from similar concentrations (5-7 µg/mL) of autoantibodies in diabetic (n=12) or non-diabetic patients without depression (n=7). Ten micromolar concentrations of Y27632, a selective Rho-Associated Protein Kinase (ROCK) inhibitor, significantly prevented (P<0.0001) neural progenitor cell process retraction induced by diabetes depression autoantibodies (n=5). Mice treated with diabetic depression autoantibodies (n=16 from two different patients' autoantibodies) exhibited significantly reduced (P=0.027) sucrose preference (anhedonia) compared to mice treated with diabetic control autoantibodies (n=16 from two different patients' autoantibodies). CONCLUSION: These data suggest that autoantibodies in a subset of older adult diabetic depression inhibit endothelial cell survival, and impair process extension and survival in adult dentate gyrus neural progenitor cells in vitro.

VGF (TLQP-62)-induced neurogenesis targets early phase neural progenitor cells in the adult hippocampus and requires glutamate and BDNF signaling.

The neuropeptide VGF (non-acronymic), which has antidepressant-like effects, enhances adult hippocampal neurogenesis as well as synaptic activity and plasticity in the hippocampus, however the interaction between these processes and the mechanism underlying this regulation remain unclear. In this study, we demonstrate that VGF-derived peptide TLQP-62 specifically enhances the generation of early progenitor cells in nestin-GFP mice. Specifically, TLQP-62 significantly increases the number of Type 2a neural progenitor cells (NPCs) while reducing the number of more differentiated Type 3 cells. The effect of TLQP-62 on proliferation rather than differentiation was confirmed using NPCs in vitro; TLQP-62 but not scrambled peptide PEHN-62 increases proliferation in a cell line as well as in primary progenitors from adult hippocampus. Moreover, TLQP-62 but not scrambled peptide increases Cyclin D mRNA expression. The proliferation of NPCs induced by TLQP-62 requires synaptic activity, in particular through NMDA and metabotropic glutamate receptors. The activation of glutamate receptors by TLQP-62 activation induces phosphorylation of CaMKII through NMDA receptors and protein kinase D through metabotropic glutamate receptor 5 (mGluR5). Furthermore, pharmacological antagonists to CaMKII and PKD inhibit TLQP-62-induced proliferation of NPCs indicating that these signaling molecules downstream of glutamate receptors are essential for the actions of TLQP-62 on neurogenesis. We also show that TLQP-62 gradually activates Brain-Derived Neurotrophic Factor (BDNF)-receptor TrkB in vitro and that Trk signaling is required for TLQP-62-induced proliferation of NPCs. Understanding the precise molecular mechanism of how TLQP-62 influences neurogenesis may reveal mechanisms by which VGF-derived peptides act as antidepressant-like agents.