Kyle A. Lyman

Chronic granulomatous disease: A large, single-center US experience

Background: Chronic granulomatous disease (CGD) is an uncommon primary immunodeficiency that can be inherited in an X-linked (XL) or an autosomal recessive (AR) manner. We reviewed our large, single-center US experience with CGD. Methods: We reviewed 27 patients at Ann & Robert H. Lurie Children’s Hospital of Chicago from March 1985 to November 2013. Fisher exact test was used to compare differences in categorical variables, and Student t test was used to compare means for continuous variables. Serious infections were defined as those requiring intravenous antibiotics or hospitalization. Results: There were 23 males and 4 females; 19 were XL and 8 were AR. The average age at diagnosis was 3.0 years; 2.1 years for XL and 5.3 years for AR inheritance (P = 0.02). There were 128 serious infections. The most frequent infectious agents were Staphylococcus aureus (n = 13), Serratia (n = 11), Klebsiella (n = 7), Aspergillus (n = 6) and Burkholderia (n = 4). The most common serious infections were pneumonia (n = 38), abscess (n = 32) and lymphadenitis (n = 29). Thirteen patients had granulomatous complications. Five patients were below the 5th percentile for height and 4 were below the 5th percentile for weight. Average length of follow-up after diagnosis was 10.1 years. Twenty-four patients were compliant and maintained on interferon-&ggr;, trimethoprim-sulfamethoxazole and an azole. The serious infection rate was 0.62 per patient-year. Twenty-three patients are alive (1 was lost to follow-up). Conclusions: We present a large, single-center US experience with CGD. Twenty-three of 27 patients are alive after 3276 patient-months of follow-up (1 has been lost to follow-up), and our serious infection rate was 0.62 per patient-year.

HCN-channel dendritic targeting requires bipartite interaction with TRIP8b and regulates antidepressant-like behavioral effects

Major depressive disorder (MDD) is a prevalent psychiatric condition with limited therapeutic options beyond monoaminergic therapies. Although effective in some individuals, many patients fail to respond adequately to existing treatments, and new pharmacologic targets are needed. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate excitability in neurons, and blocking HCN channel function has been proposed as a novel antidepressant strategy. However, systemic blockade of HCN channels produces cardiac effects that limit this approach. Knockout (KO) of the brain-specific HCN-channel auxiliary subunit tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) also produces antidepressant-like behavioral effects and suggests that inhibiting TRIP8b function could produce antidepressant-like effects without affecting the heart. We examined the structural basis of TRIP8b-mediated HCN-channel trafficking and its relationship with antidepressant-like behavior using a viral rescue approach in TRIP8b KO mice. We found that restoring TRIP8b to the hippocampus was sufficient to reverse the impaired HCN-channel trafficking and antidepressant-like behavioral effects caused by TRIP8b KO. Moreover, we found that hippocampal expression of a mutated version of TRIP8b further impaired HCN-channel trafficking and increased the antidepressant-like behavioral phenotype of TRIP8b KO mice. Thus, modulating the TRIP8b–HCN interaction bidirectionally influences channel trafficking and antidepressant-like behavior. Overall, our work suggests that small-molecule inhibitors of the interaction between TRIP8b and HCN should produce antidepressant-like behaviors and could represent a new paradigm for the treatment of MDD.

Identification of Small-Molecule Inhibitors of Hyperpolarization-Activated Cyclic Nucleotide–Gated Channels

Hyperpolarization-activated cyclic nucleotide–gated (HCN) channels function in the brain to limit neuronal excitability. Limiting the activity of these channels has been proposed as a therapy for major depressive disorder, but the critical role of HCN channels in cardiac pacemaking has limited efforts to develop therapies directed at the channel. Previous studies indicated that the function of HCN is tightly regulated by its auxiliary subunit, tetratricopeptide repeat–containing Rab8b interacting protein (TRIP8b), which is not expressed in the heart. To target the function of the HCN channel in the brain without affecting the channel’s function in the heart, we propose disrupting the interaction between HCN and TRIP8b. We developed a high-throughput fluorescence polarization (FP) assay to identify small molecules capable of disrupting this interaction. We used this FP assay to screen a 20,000-compound library and identified a number of active compounds. The active compounds were validated using an orthogonal AlphaScreen assay to identify one compound (0.005%) as the first confirmed hit for inhibiting the HCN-TRIP8b interaction. Identifying small molecules capable of disrupting the interaction between HCN and TRIP8b should enable the development of new research tools and small-molecule therapies that could benefit patients with depression.

Reduction of thalamic and cortical Ih by deletion of TRIP8b produces a mouse model of human absence epilepsy

Absence seizures occur in several types of human epilepsy and result from widespread, synchronous feedback between the cortex and thalamus that produces brief episodes of loss of consciousness. Genetic rodent models have been invaluable for investigating the pathophysiological basis of these seizures. Here, we identify tetratricopeptide-containing Rab8b-interacting protein (TRIP8b) knockout mice as a new model of absence epilepsy, featuring spontaneous spike-wave discharges on electroencephalography (EEG) that are the electrographic hallmark of absence seizures. TRIP8b is an auxiliary subunit of the hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels, which have previously been implicated in the pathogenesis of absence seizures. In contrast to mice lacking the pore-forming HCN channel subunit HCN2, TRIP8b knockout mice exhibited normal cardiac and motor function and a less severe seizure phenotype. Evaluating the circuit that underlies absence seizures, we found that TRIP8b knockout mice had significantly reduced HCN channel expression and function in thalamic-projecting cortical layer 5b neurons and thalamic relay neurons, but preserved function in inhibitory neurons of the reticular thalamic nucleus. Our results expand the known roles of TRIP8b and provide new insight into the region-specific functions of TRIP8b and HCN channels in constraining cortico-thalamo-cortical excitability.

Animal models suggest the TRIP8b-HCN interaction is a therapeutic target for major depressive disorder

Major depressive disorder (MDD) remains a substantial public health problem despite the existence of many available treatments. One issue with existing pharmacological options is that nearly all of them act to increase monoaminergic neurotransmission. Given the mechanistic similarity, it is not surprising that many patients fail to respond adequately to current treatments. Recent work using animal models of MDD has suggested that the excitability of the hippocampus may play an important role in mediating the symptoms of MDD [1]. In line with this hypothesis, human patients diagnosed with MDD show less hippocampal functional connectivity [1]. This potential MDD mechanism suggests that therapeutic interventions that increase hippocampal excitability may function as antidepressants.

Cortical compass: EML1 helps point the way in neuronal migration

Commentary Defects in neuronal migration constitute a broad class of developmental disorders that lead to the ectopic localization of neurons. In many cases, deficits in cell motility, proliferation, and surrounding parenchyma have been implicated (1). The phenotype of patients with neuronal migration defects ranges from subclinical to debilitating (2). Of particular relevance for neurologists is that although cortical malformations arising from migration disorders affect only 1% of the population, the incidence rises to 14% in patients with epilepsy (3). Subcortical band heterotopia (SBH) is often called “double cortex syndrome” (3) based on the presence of bands of ectopic gray matter within the white matter below the cortex (2). Clinically, SBH is most often caused by mutation of the gene doublecortin (DCX) that leads to moderate cognitive deficits and severe epilepsy (2). However, multiple etiologies in addition to DCX mutations also lead to SBH (1). Recently, a spontaneous mutant mouse (called the HeCo mouse for “heterotopic cortex”) was identified with a phenotype consistent with SBH, including band heterotopia, developmental delay, and lowered threshold for seizures (4). In a groundbreaking work published earlier this year in Nature Neuroscience, Kielar and colleagues set out to identify the gene responsible for the HeCo mouse phenotype as well as its function. The authors first characterized the neurons in the heterotopic band by colabeling the neurons at several time points for markers of earlyand late-born developmental identity. Surprisingly, they noted that both early and late progenitors were present in the heterotopic gray matter (although they arrive there in a delayed fashion relative to wildtype). The insight that both earlyand late-born neurons migrated to the heterotopic cortex provided the first clue that the mutation in the HeCo mice was unrelated to the capacity of the neurons to migrate. To demonstrate directly that the migratory capacity of the HeCo neurons was intact, the authors used slice cultures and labeled neurons with GFP. By video monitoring the slices, they were able to watch the HeCo neurons initial development from progenitors as well as the subsequent migration of neurons into the cortical plate. Surprisingly, they noted that the HeCo neurons migrated at the same speed as those in the wild-type animals. This is of particular relevance, as many other genetic mutations implicated in SBH cause compromised migratory capacity (1). Interestingly, despite normal migration speed, fewer HeCo neurons successfully arrived in the cortical plate (the precursor structure to the cortex in the adult). This suggested the possibility that although the HeCo neurons themselves were capable of migration, perhaps they were inappropriately responding to extracellular cues. To examine this possibility, they transplanted labeled HeCo neurons into wild-type cultures to observe their migratory capacity. Again, they saw no difference in the rate of migration of the HeCo cells. These experiments strongly suggest that the defect in HeCo mice does not affect neuronal migration. Mutations in EML1 Lead to Ectopic Progenitors and Neuronal Heterotopia in Mouse and Human.

HCN channels in the hippocampus regulate active coping behavior

Active coping is an adaptive stress response that improves outcomes in medical and neuropsychiatric diseases. To date, most research into coping style has focused on neurotransmitter activity and little is known about the intrinsic excitability of neurons in the associated brain regions that facilitate coping. Previous studies have shown that HCN channels regulate neuronal excitability in pyramidal cells and that HCN channel current (Ih) in the CA1 area increases with chronic mild stress. Reduction of Ih in the CA1 area leads to antidepressant‐like behavior, and this region has been implicated in the regulation of coping style. We hypothesized that the antidepressant‐like behavior achieved with CA1 knockdown of Ih is accompanied by increases in active coping. In this report, we found that global loss of TRIP8b, a necessary subunit for proper HCN channel localization in pyramidal cells, led to active coping behavior in numerous assays specific to coping style. We next employed a viral strategy using a dominant negative TRIP8b isoform to alter coping behavior by reducing HCN channel expression. This approach led to a robust reduction in Ih in CA1 pyramidal neurons and an increase in active coping. Together, these results establish that changes in HCN channel function in CA1 influences coping style.

Allostery between two binding sites in the ion channel subunit TRIP8b confers binding specificity to HCN channels

Tetratricopeptide repeat (TPR) domains are ubiquitous structural motifs that mediate protein–protein interactions. For example, the TPR domains in the peroxisomal import receptor PEX5 enable binding to a range of type 1 peroxisomal targeting signal motifs. A homolog of PEX5, tetratricopeptide repeat–containing Rab8b-interacting protein (TRIP8b), binds to and functions as an auxiliary subunit of hyperpolarization-activated cyclic nucleotide (HCN)–gated channels. Given the similarity between TRIP8b and PEX5, this difference in function raises the question of what mechanism accounts for their binding specificity. In this report, we found that the cyclic nucleotide–binding domain and the C terminus of the HCN channel are critical for conferring specificity to TRIP8b binding. We show that TRIP8b binds the HCN cyclic nucleotide–binding domain through a 37-residue domain and the HCN C terminus through the TPR domains. Using a combination of fluorescence polarization– and co-immunoprecipitation–based assays, we establish that binding at either site increases affinity at the other. Thus, allosteric coupling of the TRIP8b TPR domains both promotes binding to HCN channels and limits binding to type 1 peroxisomal targeting signal substrates. These results raise the possibility that other TPR domains may be similarly influenced by allosteric mechanisms as a general feature of protein–protein interactions.