Valine and Inflammation Drive Epilepsy in a Mouse Model of ECHS1 Deficiency.

ECHS1 Deficiency (ECHS1D) is a rare and devastating pediatric disease that currently has no defined treatments. This disorder results from missense loss-of-function mutations in the ECHS1 gene that result in severe developmental delays, encephalopathy, hypotonia, and early death. ECHS1 enzymatic activity is necessary for the beta-oxidation of fatty acids and the oxidation of branched-chain amino acids within the inner mitochondrial matrix. The pathogenesis of disease remains unknown, however it is hypothesized that disease is driven by an accumulation of toxic metabolites from impaired valine oxidation. To expand our knowledge on disease mechanisms, a novel mouse model of ECHS1D was generated that possesses a disease-associated knock-in (KI) allele and a knock-out (KO) allele. To investigate the behavioral phenotype, a battery of testing was performed at multiple time points, which included assessments of learning, motor function, endurance, sensory responses, and anxiety. Neurological abnormalities were assessed using wireless telemetry EEG recordings, pentylenetetrazol (PTZ) seizure induction, and immunohistochemistry. Metabolic perturbations were measured within the liver, serum, and brain using mass spectrometry and magnetic resonance spectroscopy. To test disease mechanisms, mice were subjected to disease pathway stressors and then survival, body weight gain, and epilepsy were assessed. Mice containing KI/KI or KI/KO alleles were viable with normal development and survival, and the presence of KI and KO alleles resulted in a significant reduction in ECHS1 protein. ECHS1D mice displayed reduced exercise capacity and pain sensation. EEG analysis revealed increased slow wave power that was associated with perturbations in sleep. ECHS1D mice had significantly increased epileptiform EEG discharges, and were sensitive to seizure induction, which resulted in death of 60% of ECHS1D mice. Under basal conditions, brain structure was grossly normal, although histological analysis revealed increased microglial activation in aged ECHS1D mice. Increased dietary valine only affected ECHS1D mice, which significantly exacerbated seizure susceptibility and resulted in death. Lastly, acute inflammatory challenge drove regression and early lethality in ECHS1D mice. In conclusion, we developed a novel model of ECHS1D that may be used to further knowledge on disease mechanisms and to develop therapeutics. Our data suggests altered metabolic signaling and inflammation may contribute to epilepsy in ECHS1D, and these alterations may be attributed to impaired valine metabolism.

Gain-of-function mutations of TRPV4 acting in endothelial cells drive blood-CNS barrier breakdown and motor neuron degeneration in mice.

Blood-CNS barrier disruption is a hallmark of numerous neurological disorders, yet whether barrier breakdown is sufficient to trigger neurodegenerative disease remains unresolved. Therapeutic strategies to mitigate barrier hyperpermeability are also limited. Dominant missense mutations of the cation channel transient receptor potential vanilloid 4 (TRPV4) cause forms of hereditary motor neuron disease. To gain insights into the cellular basis of these disorders, we generated knock-in mouse models of TRPV4 channelopathy by introducing two disease-causing mutations (R269C and R232C) into the endogenous mouse Trpv4 gene. TRPV4 mutant mice exhibited weakness, early lethality, and regional motor neuron loss. Genetic deletion of the mutant Trpv4 allele from endothelial cells (but not neurons, glia, or muscle) rescued these phenotypes. Symptomatic mutant mice exhibited focal disruptions of blood-spinal cord barrier (BSCB) integrity, associated with a gain of function of mutant TRPV4 channel activity in neural vascular endothelial cells (NVECs) and alterations of NVEC tight junction structure. Systemic administration of a TRPV4-specific antagonist abrogated channel-mediated BSCB impairments and provided a marked phenotypic rescue of symptomatic mutant mice. Together, our findings show that mutant TRPV4 channels can drive motor neuron degeneration in a non-cell autonomous manner by precipitating focal breakdown of the BSCB. Further, these data highlight the reversibility of TRPV4-mediated BSCB impairments and identify a potential therapeutic strategy for patients with TRPV4 mutations.

Plural molecular and cellular mechanisms of pore domain KCNQ2 encephalopathy.

KCNQ2 variants in children with neurodevelopmental impairment are difficult to assess due to their heterogeneity and unclear pathogenic mechanisms. We describe a child with neonatal-onset epilepsy, developmental impairment of intermediate severity, and KCNQ2 G256W heterozygosity. Analyzing prior KCNQ2 channel cryoelectron microscopy models revealed G256 as a node of an arch-shaped non-covalent bond network linking S5, the pore turret, and the ion path. Co-expression with G256W dominantly suppressed conduction by wild-type subunits in heterologous cells. Ezogabine partly reversed this suppression. G256W/+ mice have epilepsy leading to premature deaths. Hippocampal CA1 pyramidal cells from G256W/+ brain slices showed hyperexcitability. G256W/+ pyramidal cell KCNQ2 and KCNQ3 immunolabeling was significantly shifted from axon initial segments to neuronal somata. Despite normal mRNA levels, G256W/+ mouse KCNQ2 protein levels were reduced by about 50%. Our findings indicate that G256W pathogenicity results from multiplicative effects, including reductions in intrinsic conduction, subcellular targeting, and protein stability. These studies provide evidence for an unexpected and novel role for the KCNQ2 pore turret and introduce a valid animal model of KCNQ2 encephalopathy. Our results, spanning structure to behavior, may be broadly applicable because the majority of KCNQ2 encephalopathy patients share variants near the selectivity filter.

Stathmin-2 loss leads to neurofilament-dependent axonal collapse driving motor and sensory denervation.

The mRNA transcript of the human STMN2 gene, encoding for stathmin-2 protein (also called SCG10), is profoundly impacted by TAR DNA-binding protein 43 (TDP-43) loss of function. The latter is a hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Using a combination of approaches, including transient antisense oligonucleotide-mediated suppression, sustained shRNA-induced depletion in aging mice, and germline deletion, we show that stathmin-2 has an important role in the establishment and maintenance of neurofilament-dependent axoplasmic organization that is critical for preserving the caliber and conduction velocity of myelinated large-diameter axons. Persistent stathmin-2 loss in adult mice results in pathologies found in ALS, including reduced interneurofilament spacing, axonal caliber collapse that drives tearing within outer myelin layers, diminished conduction velocity, progressive motor and sensory deficits, and muscle denervation. These findings reinforce restoration of stathmin-2 as an attractive therapeutic approach for ALS and other TDP-43-dependent neurodegenerative diseases.

Mechanism of STMN2 cryptic splice-polyadenylation and its correction for TDP-43 proteinopathies.

Loss of nuclear TDP-43 is a hallmark of neurodegeneration in TDP-43 proteinopathies, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 mislocalization results in cryptic splicing and polyadenylation of pre-messenger RNAs (pre-mRNAs) encoding stathmin-2 (also known as SCG10), a protein that is required for axonal regeneration. We found that TDP-43 binding to a GU-rich region sterically blocked recognition of the cryptic 3' splice site in STMN2 pre-mRNA. Targeting dCasRx or antisense oligonucleotides (ASOs) suppressed cryptic splicing, which restored axonal regeneration and stathmin-2-dependent lysosome trafficking in TDP-43-deficient human motor neurons. In mice that were gene-edited to contain human STMN2 cryptic splice-polyadenylation sequences, ASO injection into cerebral spinal fluid successfully corrected Stmn2 pre-mRNA misprocessing and restored stathmin-2 expression levels independently of TDP-43 binding.

Neurobehavioral deficits of mice expressing a low level of G127V mutant frataxin.

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin (FXN). Most FRDA patients are homozygous for large expansions of GAA repeats in intron 1 of FXN, while some are compound heterozygotes with an expanded GAA tract in one allele and a missense or nonsense mutation in the other. A missense mutation, changing a glycine to valine at position 130 (G130V), is prevalent among the clinical variants. We and others have demonstrated that levels of mature FXN protein in FRDA G130V samples are reduced below those detected in samples harboring homozygous repeat expansions. Little is known regarding expression and function of endogenous FXN-G130V protein due to lack of reagents and models that can distinguish the mutant FXN protein from the wild-type FXN produced from the GAA-expanded allele. We aimed to determine the effect of the G130V (murine G127V) mutation on Fxn expression and to define its multi-system impact in vivo. We used CRISPR/Cas9 to introduce the G127V missense mutation in the Fxn coding sequence and generated homozygous mice (FxnG127V/G127V). We also introduced the G127V mutation into a GAA repeat expansion FRDA mouse model (FxnGAA230/KO; KIKO) to generate a compound heterozygous strain (FxnG127V/GAA230). We performed neurobehavioral tests on cohorts of WT and Fxn mutant animals at three-month intervals for one year, and collected tissue samples to analyze molecular changes during that time. The endogenous Fxn G127V protein is detected at much lower levels in all tissues analyzed from FxnG127V/G127V mice compared to age and sex-matched WT mice without differences in Fxn transcript levels. FxnG127V/G127V mice are significantly smaller than WT counterparts, but perform similarly in most neurobehavioral tasks. RNA sequencing analysis revealed reduced expression of genes in oxidative phosphorylation and protein synthesis, underscoring the metabolic consequences in our mouse model expressing extremely low levels of Fxn. Results of these studies provide insight into the unique pathogenic mechanism of the FXN G130V mechanism and the tolerable limit of Fxn/FXN expression in vivo.

MATR3 P154S knock-in mice do not exhibit motor, muscle or neuropathologic features of ALS.

Matrin 3 is a nuclear matrix protein that has many roles in RNA processing including splicing and transport of mRNA. Many missense mutations in the Matrin 3 gene (MATR3) have been linked to familial forms of amyotrophic lateral sclerosis (ALS) and distal myopathy. However, the exact role of MATR3 mutations in ALS and myopathy pathogenesis is not understood. To demonstrate a role of MATR3 mutations in vivo, we generated a novel CRISPR/Cas9 mediated knock-in mouse model harboring the MATR3 P154S mutation expressed under the control of the endogenous promoter. The P154S variant of the MATR3 gene has been linked to familial forms of ALS. Heterozygous and homozygous MATR3 P154S knock-in mice did not develop progressive motor deficits compared to wild-type mice. In addition, ALS-like pathology did not develop in nervous or muscle tissue in either heterozygous or homozygous mice. Our results suggest that the MATR3 P154S variant is not sufficient to produce ALS-like pathology in vivo.

New mouse models with hypomorphic SUMF1 variants mimic attenuated forms of multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD) is an ultrarare lysosomal storage disorder due to deficiency of all known sulfatases. MSD is caused by mutations in the Sulfatase Modifying Factor 1 (SUMF1) gene encoding the enzyme responsible for the post-translational modification and activation of all sulfatases. Most MSD patients carry hypomorph SUMF1 variants resulting in variable degrees of residual sulfatase activities. In contrast, Sumf1 null mice with complete deficiency in all sulfatase enzyme activities, have very short lifespan with significant pre-wean lethality, owing to a challenging preclinical model. To overcome this limitation, we genetically engineered and characterized in mice two commonly identified patient-based SUMF1 pathogenic variants, namely p.Ser153Pro and p.Ala277Val. These pathogenic missense variants correspond to variants detected in patients with attenuated MSD presenting with partial-enzyme deficiency and relatively less severe disease. These novel MSD mouse models have a longer lifespan and show biochemical and pathological abnormalities observed in humans. In conclusion, mice harboring the p.Ser153Pro or the p.Ala277Val variant mimic the attenuated MSD and are attractive preclinical models for investigation of pathogenesis and treatments for MSD.

Loss of Endothelial TDP-43 Leads to Blood Brain Barrier Defects in Mouse Models of Amyotrophic Lateral Sclerosis and Frontotemporal Dementia.

Loss of nuclear TDP-43 occurs in a wide range of neurodegenerative diseases, and specific mutations in the TARDBP gene that encodes the protein are linked to familial Frontal Temporal Lobar Dementia (FTD), and Amyotrophic Lateral Sclerosis (ALS). Although the focus has been on neuronal cell dysfunction caused by TDP-43 variants, TARDBP mRNA transcripts are expressed at similar levels in brain endothelial cells (ECs). Since increased permeability across the blood brain barrier (BBB) precedes cognitive decline, we postulated that altered functions of TDP-43 in ECs contributes to BBB dysfunction in neurodegenerative disease. To test this hypothesis, we examined EC function and BBB properties in mice with either knock-in mutations found in ALS/FTLD patients (TARDBPG348C and GRNR493X) or EC-specific deletion of TDP-43 throughout the endothelium (Cdh5(PAC)CreERT2; Tardbpff) or restricted to brain endothelium (Slco1c1(BAC)CreERT2; Tardbpff). We found that TARDBPG348C mice exhibited increased permeability to 3kDa Texas Red dextran and NHS-biotin, relative to their littermate controls, which could be recapitulated in cultured brain ECs from these mice. Nuclear levels of TDP-43 were reduced in vitro and in vivo in ECs from TARDBPG348C mice. This coincided with a reduction in junctional proteins VE-cadherin, claudin-5 and ZO-1 in isolated ECs, supporting a cell autonomous effect on barrier function through a loss of nuclear TDP-43. We further examined two models of Tardbp deletion in ECs, and found that the loss of TDP-43 throughout the endothelium led to systemic endothelial activation and permeability. Deletion specifically within the brain endothelium acutely increased BBB permeability, and eventually led to hallmarks of FTD, including fibrin deposition, microglial and astrocyte activation, and behavioral defects. Together, these data show that TDP-43 dysfunction specifically within brain ECs would contribute to the BBB defects observed early in the progression of ALS/FTLD.

Loss of Pex1 in Inner Ear Hair Cells Contributes to Cochlear Synaptopathy and Hearing Loss.

Peroxisome Biogenesis Disorders (PBD) and Zellweger syndrome spectrum disorders (ZSD) are rare genetic multisystem disorders that include hearing impairment and are associated with defects in peroxisome assembly, function, or both. Mutations in 13 peroxin (PEX) genes have been found to cause PBD-ZSD with ~70% of patients harboring mutations in PEX1. Limited research has focused on the impact of peroxisomal disorders on auditory function. As sensory hair cells are particularly vulnerable to metabolic changes, we hypothesize that mutations in PEX1 lead to oxidative stress affecting hair cells of the inner ear, subsequently resulting in hair cell degeneration and hearing loss. Global deletion of the Pex1 gene is neonatal lethal in mice, impairing any postnatal studies. To overcome this limitation, we created conditional knockout mice (cKO) using Gfi1Creor VGlut3Cre expressing mice crossed to floxed Pex1 mice to allow for selective deletion of Pex1 in the hair cells of the inner ear. We find that Pex1 excision in inner hair cells (IHCs) leads to progressive hearing loss associated with significant decrease in auditory brainstem responses (ABR), specifically ABR wave I amplitude, indicative of synaptic defects. Analysis of IHC synapses in cKO mice reveals a decrease in ribbon synapse volume and functional alterations in exocytosis. Concomitantly, we observe a decrease in peroxisomal number, indicative of oxidative stress imbalance. Taken together, these results suggest a critical function of Pex1 in development and maturation of IHC-spiral ganglion synapses and auditory function.

A Drosophila screen identifies NKCC1 as a modifier of NGLY1 deficiency.

N-Glycanase 1 (NGLY1) is a cytoplasmic deglycosylating enzyme. Loss-of-function mutations in the NGLY1 gene cause NGLY1 deficiency, which is characterized by developmental delay, seizures, and a lack of sweat and tears. To model the phenotypic variability observed among patients, we crossed a Drosophila model of NGLY1 deficiency onto a panel of genetically diverse strains. The resulting progeny showed a phenotypic spectrum from 0 to 100% lethality. Association analysis on the lethality phenotype, as well as an evolutionary rate covariation analysis, generated lists of modifying genes, providing insight into NGLY1 function and disease. The top association hit was Ncc69 (human NKCC1/2), a conserved ion transporter. Analyses in NGLY1-/- mouse cells demonstrated that NKCC1 has an altered average molecular weight and reduced function. The misregulation of this ion transporter may explain the observed defects in secretory epithelium function in NGLY1 deficiency patients.

Loss of Tmem106b exacerbates FTLD pathologies and causes motor deficits in progranulin-deficient mice.

Progranulin (PGRN) and transmembrane protein 106B (TMEM106B) are important lysosomal proteins implicated in frontotemporal lobar degeneration (FTLD) and other neurodegenerative disorders. Loss-of-function mutations in progranulin (GRN) are a common cause of FTLD, while TMEM106B variants have been shown to act as disease modifiers in FTLD. Overexpression of TMEM106B leads to lysosomal dysfunction, while loss of Tmem106b ameliorates lysosomal and FTLD-related pathologies in young Grn-/- mice, suggesting that lowering TMEM106B might be an attractive strategy for therapeutic treatment of FTLD-GRN. Here, we generate and characterize older Tmem106b-/- Grn-/- double knockout mice, which unexpectedly show severe motor deficits and spinal cord motor neuron and myelin loss, leading to paralysis and premature death at 11-12 months. Compared to Grn-/- , Tmem106b-/- Grn-/- mice have exacerbated FTLD-related pathologies, including microgliosis, astrogliosis, ubiquitin, and phospho-Tdp43 inclusions, as well as worsening of lysosomal and autophagic deficits. Our findings confirm a functional interaction between Tmem106b and Pgrn and underscore the need to rethink whether modulating TMEM106B levels is a viable therapeutic strategy.

Regulation of BMP4/Dpp retrotranslocation and signaling by deglycosylation.

During endoplasmic reticulum-associated degradation (ERAD), the cytoplasmic enzyme N-glycanase 1 (NGLY1) is proposed to remove N-glycans from misfolded N-glycoproteins after their retrotranslocation from the ER to the cytosol. We previously reported that NGLY1 regulates Drosophila BMP signaling in a tissue-specific manner (Galeone et al., 2017). Here, we establish the Drosophila Dpp and its mouse ortholog BMP4 as biologically relevant targets of NGLY1 and find, unexpectedly, that NGLY1-mediated deglycosylation of misfolded BMP4 is required for its retrotranslocation. Accumulation of misfolded BMP4 in the ER results in ER stress and prompts the ER recruitment of NGLY1. The ER-associated NGLY1 then deglycosylates misfolded BMP4 molecules to promote their retrotranslocation and proteasomal degradation, thereby allowing properly-folded BMP4 molecules to proceed through the secretory pathway and activate signaling in other cells. Our study redefines the role of NGLY1 during ERAD and suggests that impaired BMP4 signaling might underlie some of the NGLY1 deficiency patient phenotypes.

Loss of TMEM106B leads to myelination deficits: implications for frontotemporal dementia treatment strategies.

Genetic variants that define two distinct haplotypes at the TMEM106B locus have been implicated in multiple neurodegenerative diseases and in healthy brain ageing. In frontotemporal dementia (FTD), the high expressing TMEM106B risk haplotype was shown to increase susceptibility for FTD with TDP-43 inclusions (FTD-TDP) and to modify disease penetrance in progranulin mutation carriers (FTD-GRN). To elucidate the biological function of TMEM106B and determine whether lowering TMEM106B may be a viable therapeutic strategy, we performed brain transcriptomic analyses in 8-month-old animals from our recently developed Tmem106b-/- mouse model. We included 10 Tmem106b+/+ (wild-type), 10 Tmem106b+/- and 10 Tmem106-/- mice. The most differentially expressed genes (153 downregulated and 60 upregulated) were identified between Tmem106b-/- and wild-type animals, with an enrichment for genes implicated in myelination-related cellular processes including axon ensheathment and oligodendrocyte differentiation. Co-expression analysis also revealed that the most downregulated group of correlated genes was enriched for myelination-related processes. We further detected a significant loss of OLIG2-positive cells in the corpus callosum of Tmem106b-/- mice, which was present already in young animals (21 days) and persisted until old age (23 months), without worsening. Quantitative polymerase chain reaction revealed a reduction of differentiated but not undifferentiated oligodendrocytes cellular markers. While no obvious changes in myelin were observed at the ultrastructure levels in unchallenged animals, treatment with cuprizone revealed that Tmem106b-/- mice are more susceptible to cuprizone-induced demyelination and have a reduced capacity to remyelinate, a finding which we were able to replicate in a newly generated Tmem106b CRISPR/cas9 knock-out mouse model. Finally, using a TMEM106B HeLa knock-out cell line and primary cultured oligodendrocytes, we determined that loss of TMEM106B leads to abnormalities in the distribution of lysosomes and PLP1. Together these findings reveal an important function for TMEM106B in myelination with possible consequences for therapeutic strategies aimed at lowering TMEM106B levels.

Mitochondrial damage and senescence phenotype of cells derived from a novel frataxin G127V point mutation mouse model of Friedreich's ataxia.

Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin (FXN). Most FRDA patients are homozygous for large expansions of GAA repeat sequences in intron 1 of FXN, whereas a fraction of patients are compound heterozygotes, with a missense or nonsense mutation in one FXN allele and expanded GAAs in the other. A prevalent missense mutation among FRDA patients changes a glycine at position 130 to valine (G130V). Herein, we report generation of the first mouse model harboring an Fxn point mutation. Changing the evolutionarily conserved glycine 127 in mouse Fxn to valine results in a failure-to-thrive phenotype in homozygous animals and a substantially reduced number of offspring. Like G130V in FRDA, the G127V mutation results in a dramatic decrease of Fxn protein without affecting transcript synthesis or splicing. FxnG127V mouse embryonic fibroblasts exhibit significantly reduced proliferation and increased cell senescence. These defects are evident in early passage cells and are exacerbated at later passages. Furthermore, increased frequency of mitochondrial DNA lesions and fragmentation are accompanied by marked amplification of mitochondrial DNA in FxnG127V cells. Bioenergetics analyses demonstrate higher sensitivity and reduced cellular respiration of FxnG127V cells upon alteration of fatty acid availability. Importantly, substitution of FxnWT with FxnG127V is compatible with life, and cellular proliferation defects can be rescued by mitigation of oxidative stress via hypoxia or induction of the NRF2 pathway. We propose FxnG127V cells as a simple and robust model for testing therapeutic approaches for FRDA.

Dominant mutations of the Notch ligand Jagged1 cause peripheral neuropathy.

Notch signaling is a highly conserved intercellular pathway with tightly regulated and pleiotropic roles in normal tissue development and homeostasis. Dysregulated Notch signaling has also been implicated in human disease, including multiple forms of cancer, and represents an emerging therapeutic target. Successful development of such therapeutics requires a detailed understanding of potential on-target toxicities. Here, we identify autosomal dominant mutations of the canonical Notch ligand Jagged1 (or JAG1) as a cause of peripheral nerve disease in 2 unrelated families with the hereditary axonal neuropathy Charcot-Marie-Tooth disease type 2 (CMT2). Affected individuals in both families exhibited severe vocal fold paresis, a rare feature of peripheral nerve disease that can be life-threatening. Our studies of mutant protein posttranslational modification and localization indicated that the mutations (p.Ser577Arg, p.Ser650Pro) impair protein glycosylation and reduce JAG1 cell surface expression. Mice harboring heterozygous CMT2-associated mutations exhibited mild peripheral neuropathy, and homozygous expression resulted in embryonic lethality by midgestation. Together, our findings highlight a critical role for JAG1 in maintaining peripheral nerve integrity, particularly in the recurrent laryngeal nerve, and provide a basis for the evaluation of peripheral neuropathy as part of the clinical development of Notch pathway-modulating therapeutics.

ALS/FTD mutant CHCHD10 mice reveal a tissue-specific toxic gain-of-function and mitochondrial stress response.

Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10), a mitochondrial protein of unknown function, cause a disease spectrum with clinical features of motor neuron disease, dementia, myopathy and cardiomyopathy. To investigate the pathogenic mechanisms of CHCHD10, we generated mutant knock-in mice harboring the mouse-equivalent of a disease-associated human S59L mutation, S55L in the endogenous mouse gene. CHCHD10S55L mice develop progressive motor deficits, myopathy, cardiomyopathy and accelerated mortality. Critically, CHCHD10 accumulates in aggregates with its paralog CHCHD2 specifically in affected tissues of CHCHD10S55L mice, leading to aberrant organelle morphology and function. Aggregates induce a potent mitochondrial integrated stress response (mtISR) through mTORC1 activation, with elevation of stress-induced transcription factors, secretion of myokines, upregulated serine and one-carbon metabolism, and downregulation of respiratory chain enzymes. Conversely, CHCHD10 ablation does not induce disease pathology or activate the mtISR, indicating that CHCHD10S55L-dependent disease pathology is not caused by loss-of-function. Overall, CHCHD10S55L mice recapitulate crucial aspects of human disease and reveal a novel toxic gain-of-function mechanism through maladaptive mtISR and metabolic dysregulation.

Mouse Models for Drug Discovery. Can New Tools and Technology Improve Translational Power?

The use of mouse models in biomedical research and preclinical drug evaluation is on the rise. The advent of new molecular genome-altering technologies such as CRISPR/Cas9 allows for genetic mutations to be introduced into the germ line of a mouse faster and less expensively than previous methods. In addition, the rapid progress in the development and use of somatic transgenesis using viral vectors, as well as manipulations of gene expression with siRNAs and antisense oligonucleotides, allow for even greater exploration into genomics and systems biology. These technological advances come at a time when cost reductions in genome sequencing have led to the identification of pathogenic mutations in patient populations, providing unprecedented opportunities in the use of mice to model human disease. The ease of genetic engineering in mice also offers a potential paradigm shift in resource sharing and the speed by which models are made available in the public domain. Predictively, the knowledge alone that a model can be quickly remade will provide relief to resources encumbered by licensing and Material Transfer Agreements. For decades, mouse strains have provided an exquisite experimental tool to study the pathophysiology of the disease and assess therapeutic options in a genetically defined system. However, a major limitation of the mouse has been the limited genetic diversity associated with common laboratory mice. This has been overcome with the recent development of the Collaborative Cross and Diversity Outbred mice. These strains provide new tools capable of replicating genetic diversity to that approaching the diversity found in human populations. The Collaborative Cross and Diversity Outbred strains thus provide a means to observe and characterize toxicity or efficacy of new therapeutic drugs for a given population. The combination of traditional and contemporary mouse genome editing tools, along with the addition of genetic diversity in new modeling systems, are synergistic and serve to make the mouse a better model for biomedical research, enhancing the potential for preclinical drug discovery and personalized medicine.

Variations in the contents of gingerols and chromatographic fingerprints of ginger root extracts prepared by different preparation methods.

In the present study, an HPLC-DAD method was optimized for the quantitative determination of 6-gingerol, 6-shogaol, 8-gingerol, and 10-gingerol in ginger extracts. A chromatographic fingerprinting method was also established to differentiate and evaluate the ginger extracts for bioactivity. Twenty-one extracts were prepared by methods differing in ginger type (fresh versus dried), solvent, and extraction methods. The ANOVA analysis showed the methods' influence on the mean extraction yields of gingerols increased in the order of: high pressure-high temperature (HP)>blender (BD)>low pressure (LP). The optimal solvent to extract gingerols was found to be 95% ethanol. The type of ginger used had significant effects on the content of gingerols, but its overall influence depended on the solvent used. In order to maximize the extraction efficiency of gingerols, a combination of dry ginger, 95% ethanol, and the HP extraction method should be employed. The chromatographic fingerprints were obtained to differentiate the unknown components from all ginger extracts. The similarity of the chromatographic fingerprints was used to evaluate the differences among all extracts. It can be concluded that the chromatographic fingerprints are able to ensure the stability of each extract and have some correlation with the observed bioactivity.

Hypothalamic mitochondrial dysfunction associated with anorexia in the anx/anx mouse.

The anorectic anx/anx mouse exhibits disturbed feeding behavior and aberrances, including neurodegeneration, in peptidergic neurons in the appetite regulating hypothalamic arcuate nucleus. Poor feeding in infants, as well as neurodegeneration, are common phenotypes in human disorders caused by dysfunction of the mitochondrial oxidative phosphorylation system (OXPHOS). We therefore hypothesized that the anorexia and degenerative phenotypes in the anx/anx mouse could be related to defects in the OXPHOS. In this study, we found reduced efficiency of hypothalamic OXPHOS complex I assembly and activity in the anx/anx mouse. We also recorded signs of increased oxidative stress in anx/anx hypothalamus, possibly as an effect of the decreased hypothalamic levels of fully assembled complex I, that were demonstrated by native Western blots. Furthermore, the Ndufaf1 gene, encoding a complex I assembly factor, was genetically mapped to the anx interval and found to be down-regulated in anx/anx mice. These results suggest that the anorexia and hypothalamic neurodegeneration of the anx/anx mouse are associated with dysfunction of mitochondrial complex I.