Vivian Hook

The granin gene family of neuropeptides functions as peptide neurotransmitters in the brain for the regulation of neural functions that regulate behaviors. Granins are involved in regulating cognition, memory, depression, aggression, stress, energy expenditure, inflammation, and related. Development of the human brain involves formation of synapses and their spectrum of neurotransmitters to establish neural connections that are required for brain functions. Therefore, the goal of this study was to analyze the gene expression profiles of the granin neurotransmitter genes during human brain development at prenatal, infancy, childhood, adolescence, and adult stages. Granin gene expression in brain development was assessed by quantitative RNA sequencing data from the Allen Human Brain Atlas resource. VGF (neurosecretory protein VGF) expression was significantly increased during development during the prenatal to childhood through adult stages in the anterior cingulate cortex, dorsolateral prefrontal cortex, inferolateral temporal cortex, orbital frontal cortex, posteroventral parietal cortex, primary somatosensory cortex, and primary visual cortex regions. SCG2 (secretogranin 2) expression was also significantly increased from prenatal to infancy through adult stages in anterior cingulate cortex, dorsolateral prefrontal cortex, inferolateral temporal cortex, orbital frontal cortex, posterior superior temporal cortex, posteroventral parietal cortex, primary somatosensory cortex, and primary visual cortex. A modest number of brain regions showed increased CHGA , CHGB , and SCG3 expression in the postnatal periods compared to the prenatal periods. Further, the SCG5, PCSK1N, and GNAS genes displayed minimal changes throughout development. Overall, these results demonstrate developmental upregulation of VGF and SCG2 genes, with lesser upregulation of CHGA , CHGB , and SCG3 genes, and almost no changes in SCG5 , PCSK1N , and GNAS genes during development. These findings illustrate the differential regulation of granin genes during human brain development.

Ongoing Alzheimer's disease (AD) drug development research addresses the need for therapeutic agents that can ameliorate cognitive symptoms and attenuate the course of AD synaptic deficits and neurodegeneration. There is growing interest in evaluating FDA-approved drugs for repurposing as candidate AD therapeutics. Such drugs have the advantage that data are available about their pharmaceutical properties, including doses, pharmacokinetics, pharmacodynamics, biomarkers, metabolism, and safety, to inform the design of clinical drug trials. Importantly, the suitability of such drugs with properties needed for AD requires evaluation. In the early stage of AD, degeneration of the locus coeruleus (LC) brain region results in the reduction of noradrenergic neurons and the loss of the neurotransmitter norepinephrine (NE) that regulates cognition and degeneration. Elevation of extracellular NE through inhibition of the NE transporter (NET) is hypothesized to ameliorate AD deficits. Notably, the NET reuptake inhibitor atomoxetine, an FDA-approved drug for the treatment of attention deficit hyperactivity disorder (ADHD), provides an attractive candidate as an AD therapeutic agent because it may attenuate cognitive decline in AD patients, positively impact AD biomarkers, and reduce neuropathology. The goal of this review is to assess atomoxetine for repurposing in AD based on its ability to improve cognition, regulate NE, impact AD biomarkers, and preserve LC neuronal function, with suitable pharmacokinetics, drug metabolism, and safety based on analysis of clinical and preclinical studies. Evidence for neuroprotective effects of atomoxetine in the early stage of AD at clinically safe doses with suitable pharmaceutical properties supports its candidacy as a repurposed drug for AD therapeutics.

Background: Human Huntington's disease (HD) is a genetic neurodegenerative disorder caused by the mutant HTT gene containing CAG repeat expansions, resulting in motor dysfunction and behavioral deficits. CAG repeats of 40–53 occur in adult HD and 60–120 repeats occur in early onset juvenile HD, differing from the normal range of 5–35 repeats. Objective: The HTT gene is translated to the huntingtin (HTT) protein that interacts with proteins in the development of HD. There have been few studies of HTT protein interactors in human HD brain. Therefore, this study evaluated the hypothesis that dysregulation of HTT protein interactors occurs in human juvenile HD brains. Methods: The strategy of this study was to analyze proteomic data of human juvenile HD brain putamen and cortex regions for dysregulation of HTT interacting proteins, using a database that we compiled of HTT interactors identified in HD model systems from yeast to HD mice. Results: Results showed significant dysregulation of HTT protein interactors of mitochondria, signal transduction, RNA splicing, chromatin organization, translation, membrane trafficking, endocytosis, vesicle, protein modification, granule membrane, and macroautophagy pathways. The majority of downregulated and upregulated HTT interactors occurred in the putamen region compared to cortex. Dysregulation displayed downregulation of mitochondria and signal transduction interactors, combined with upregulation of RNA splicing, chromatin organization, and translational interactors. Network analysis revealed interactions among clusters of HTT interactors. Conclusions: These findings demonstrate prevalent dysregulation of HTT protein interactors in human juvenile HD brain, especially in the putamen region that controls movement deficits in HD.

Cathepsin B contributes to the behavioral deficits and neuropathology that occur in traumatic brain injury (TBI) and Alzheimer's disease (AD). TBI and AD patients display elevated levels of cathepsin B that correlate with the severity of injury or cognitive deficits, respectively. In animal models of TBI and AD, cathepsin B gene knockout ameliorates behavioral deficits and improves neuropathology. While cathepsin B is normally located in acidic lysosomes, during TBI and AD, lysosomal leakage results in the translocation of cathepsin B to the neutral pH environment of the cytosol, thereby initiating neurodegeneration. Neutral pH-selective inhibitors are hypothesized to specifically target the pathogenic cytosolic cathepsin B without affecting its normal lysosomal form. Therefore, this review focuses on a novel strategy to utilize pH-dependent substrate cleavage properties of cathepsin B for the design of a neutral pH-selective inhibitor. Investigation of the enzymatic properties of cathepsin B at different pH conditions led to the development of Z-Arg-Lys-AOMK, a neutral pH-selective inhibitor that does not affect the enzyme's activity at normal lysosomal acidic pH. Z-Arg-Lys-AOMK potently inhibits cathepsin B at nM concentrations and effectively inhibits cellular cathepsin B in neuronal cell cultures at similar levels. In mice subjected to controlled cortical impact (CCI) brain injury, a model of TBI, cytosolic cathepsin B activity was significantly elevated in the brain. Treatment of the CCI-TBI mice with Z-Arg-Lys-AOMK reduced cytosolic cathepsin B activity and resulted in less motor dysfunction. These findings show that pH-dependent cleavage properties of cathepsin B can be utilized for the development of selective inhibitors to target the neutral cytosolic form of cathepsin B. The new concept of pH-selective inhibitors of cathepsin B reveals novel opportunities for targeting pathogenic, cytosolic cathepsin B involved in brain disorders.

Cathepsin B has been shown to contribute to deficits in traumatic brain injury (TBI), an important risk factor for Alzheimer's disease (AD). Cathepsin B is elevated in TBI and AD patients, as well as in animal models of these conditions. Knockout of the cathepsin B gene results in amelioration of TBI-induced motor dysfunction and improvement of AD memory deficit in mice. The mechanism of cathepsin B pathogenesis in these brain disorders has been hypothesized to involve its translocation to the cytosol from its normal lysosomal location. This study, therefore, evaluated brain cytosolic cathepsin B activity in the controlled cortical impact (CCI) mouse model of TBI. CCI-TBI resulted in motor deficits demonstrated by the rotarod assay, brain tissue lesions, and disorganization of the hippocampus. Significantly, CCI-TBI increased cytosolic cathepsin B activity in the brain cortex in the ipsilateral brain hemisphere that received the CCI-TBI injury, with a concomitant decrease in the lysosomal fraction. Cathepsin B activity was monitored using the substrate Z-Nle-Lys-Arg-AMC which specifically detects cathepsin B activity but not other cysteine proteases. The normal lysosomal distribution of cathepsin B was observed by its discrete localization in brain cortical cells. CCI-TBI resulted in a more diffuse cellular distribution of cathepsin B consistent with translocation to the cytosol. Further studies utilized the novel neutral pH-selective inhibitor, Z-Arg-Lys-AOMK, that specifically inhibits cathepsin B at neutral pH 7.2 of the cytosol but not at acidic pH 4.6 of lysosomes. Daily administration of Z-Arg-Lys-AOMK (ip), beginning 1 day before CCI-TBI, resulted in the reduction of the increased cytosolic cathepsin B activity induced by CCI-TBI. The inhibitor also reduced cathepsin B activities in homogenates of the brain cortex and hippocampus which were increased by CCI-TBI. Furthermore, the Z-Arg-Lys-AOMK inhibitor resulted in the reduction of motor function deficit resulting from CCI-TBI. These findings demonstrate the activation of cytosolic cathepsin B activity in CCI-TBI mouse brain injury.

Therapeutic research and development for Alzheimer's disease (AD) has been an area of intense research to alleviate memory loss and neurodegeneration. There is growing interest in drug repositioning and repurposing strategies for FDA-approved medications as potential candidates that may further advance AD therapeutics. The FDA drug efavirenz has been investigated as a candidate drug for repurposing as an AD medication. The proposed mechanism of action of efavirenz (at low doses) is the activation of the neuron-specific enzyme CYP46A1 that converts excess brain cholesterol into 24-hydroxycholesterol (24-HC) that is exported to the periphery. Efavirenz at a low dose was found to improve memory deficit in the 5XFAD model of AD that was accompanied by elevated 24-HC and reduction in Aβ; furthermore, efavirenz reduced pTau and excess cholesterol levels in human iPSC-derived Alzheimer's neurons. The low dose of efavirenz used in the AD mouse model to increase 24-HC contrasts with the use of more than 100-fold higher doses of efavirenz for clinical treatment of human immunodeficiency virus (HIV) through inhibition of reverse transcriptase. Low doses of efavirenz may avoid neurotoxic adverse effects that occur at high efavirenz doses used for HIV treatment. This review evaluates the drug properties of efavirenz with respect to its preclinical data on regulating memory deficit, pharmacokinetics, pharmacodynamics, metabolites, and genetic variabilities in drug metabolism as well as its potential adverse effects. These analyses discuss the challenges and questions that should be addressed in future studies to consider the opportunity for low dose efavirenz as a candidate for AD drug development.

Schizophrenia (SZ) is a serious mental illness and neuropsychiatric brain disorder with behavioral symptoms that include hallucinations, delusions, disorganized behavior, and cognitive impairment. Regulation of such behaviors requires utilization of neurotransmitters released to mediate cell-cell communication which are essential to brain functions in health and disease. We hypothesized that SZ may involve dysregulation of neurotransmitters secreted from neurons. To gain an understanding of human SZ, induced neurons (iNs) were derived from SZ patients and healthy control subjects to investigate peptide neurotransmitters, known as neuropeptides, which represent the major class of transmitters. The iNs were subjected to depolarization by high KCl in the culture medium and the secreted neuropeptides were identified and quantitated by nano-LC-MS/MS tandem mass spectrometry. Several neuropeptides were identified from schizophrenia patient-derived neurons, including chromogranin B (CHGB), neurotensin, and natriuretic peptide. Focusing on the main secreted CHGB neuropeptides, results revealed differences in SZ iNs compared to control iN neurons. Lower numbers of distinct CHGB peptides were found in the SZ secretion media compared to controls. Mapping of the peptides to the CHGB precursor revealed peptides unique to either SZ or control, and peptides common to both conditions. Also, the iNs secreted neuropeptides under both KCl and basal (no KCl) conditions. These findings are consistent with reports that chromogranin B levels are reduced in the cerebrospinal fluid and specific brain regions of SZ patients. These findings suggest that iNs derived from SZ patients can model the decreased CHGB neuropeptides observed in human SZ.

Protecting groups (PGs) in peptide synthesis have inspired advanced design principles that incorporate "orthogonality" for selective C- and N-terminus and side-chain deprotections. The conventionally acid-stable 9-fluorenylmethoxycarbonyl (Fmoc) group is one of the most widely used N-protection groups in solid- and solution-phase synthesis. Despite the versatility of Fmoc, deprotection by the removal of the Fmoc group to unmask primary amines requires the use of a basic secondary amine nucleophile, but this stratagem poses challenges in sensitive molecules that bear reactive electrophilic groups. An expansion of PG versatility, a tunable orthogonality, in the late-stage synthesis of peptides would add flexibility to the synthetic design and implementation. Here, we report a novel Fmoc deprotection method using hydrogenolysis under mildly acidic conditions for the synthesis of Z-Arg-Lys-acyloxymethyl ketone (Z-R-K-AOMK). This new method is not only valuable for Fmoc deprotection in the synthesis of complex peptides that contain highly reactive electrophiles, or other similar sensitive functional groups, that are incompatible with traditional Fmoc deprotection conditions but also tolerant of N-Boc groups present in the substrate.

Tau propagation, pathogenesis, and neurotoxicity are hallmarks of neurodegenerative diseases that result in cognitive impairment. Tau accumulates in Alzheimer’s disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy, and related tauopathies. Knowledge of the mechanisms for tau propagation in neurodegeneration is necessary for understanding the development of dementia. Exosomes, known as extracellular vesicles (EVs), have emerged as participants in promoting tau propagation. Recent findings show that EVs generated by neurons expressing familial mutations of tauopathies of FTDP-17 (P301L and V337M) (mTau) and presenilin (A246E) (mPS1) in AD induce tau propagation and accumulation after injection into rodent brain. To gain knowledge of the proteome cargoes of the mTau and mPS1 EVs that promote tau pathogenesis, this review compares the proteomes of these EVs, which results in important new questions concerning EV mechanisms of tau pathogenesis. Proteomics data show that EVs produced by mTau- and mPS1-expressing iPSC neurons share proteins involved in exocytosis and vesicle secretion and, notably, these EVs also possess differences in protein components of vesicle-mediated transport, extracellular functions, and cell adhesion. It will be important for future studies to gain an understanding of the breadth of familial genetic mutations of tau, presenilin, and other genes in promoting EV initiation of tau propagation and pathogenesis. Furthermore, elucidation of EV cargo components that mediate tau propagation will have potential as biomarkers and therapeutic strategies to ameliorate dementia of tauopathies.

Therapeutics discovery and development for Alzheimer’s disease (AD) has been an area of intense research to alleviate memory loss and the underlying pathogenic processes. Recent drug discovery approaches have utilized in silico computational strategies for drug candidate selection which has opened the door to repurposing drugs for AD. Computational analysis of gene expression signatures of patients stratified by the APOE4 risk allele of AD led to the discovery of the FDA-approved drug bumetanide as a top candidate agent that reverses APOE4 transcriptomic brain signatures and improves memory deficits in APOE4 animal models of AD. Bumetanide is a loop diuretic which inhibits the kidney Na⁺-K⁺-2Cl⁻ cotransporter isoform, NKCC2, for the treatment of hypertension and edema in cardiovascular, liver, and renal disease. Electronic health record data revealed that patients exposed to bumetanide have lower incidences of AD by 35%–70%. In the brain, bumetanide has been proposed to antagonize the NKCC1 isoform which mediates cellular uptake of chloride ions. Blocking neuronal NKCC1 leads to a decrease in intracellular chloride and thus promotes GABAergic receptor mediated hyperpolarization, which may ameliorate disease conditions associated with GABAergic-mediated depolarization. NKCC1 is expressed in neurons and in all brain cells including glia (oligodendrocytes, microglia, and astrocytes) and the vasculature. In consideration of bumetanide as a repurposed drug for AD, this review evaluates its pharmaceutical properties with respect to its estimated brain levels across doses that can improve neurologic disease deficits of animal models to distinguish between NKCC1 and non-NKCC1 mechanisms. The available data indicate that bumetanide efficacy may occur at brain drug levels that are below those required for inhibition of the NKCC1 transporter which implicates non-NKCC1 brain mechansims for improvement of brain dysfunctions and memory deficits. Alternatively, peripheral bumetanide mechanisms may involve cells outside the central nervous system (e.g., in epithelia and the immune system). Clinical bumetanide doses for improved neurological deficits are reviewed. Regardless of mechanism, the efficacy of bumetanide to improve memory deficits in the APOE4 model of AD and its potential to reduce the incidence of AD provide support for clinical investigation of bumetanide as a repurposed AD therapeutic agent.