Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and Quantity of CRY1.

To conduct comprehensive characterization of molecular properties in organisms, we established an efficient method to produce knockout (KO)-rescue mice within a single generation. We applied this method to produce 20 strains of almost completely embryonic stem cell (ESC)-derived mice ("ES mice") rescued with wild-type and mutant Cry1 gene under a Cry1-/-:Cry2-/- background. A series of both phosphorylation-mimetic and non-phosphorylation-mimetic CRY1 mutants revealed that multisite phosphorylation of CRY1 can serve as a cumulative timer in the mammalian circadian clock. KO-rescue ES mice also revealed that CRY1-PER2 interaction confers a robust circadian rhythmicity in mice. Surprisingly, in contrast to theoretical predictions from canonical transcription/translation feedback loops, the residues surrounding the flexible P loop and C-lid domains of CRY1 determine circadian period without changing the degradation rate of CRY1. These results suggest that CRY1 determines circadian period through both its degradation-dependent and -independent pathways.

Intravenous treatment with a molecular chaperone designed against ß-amyloid toxicity improves Alzheimer's disease pathology in mouse models.

Attempts to treat Alzheimer's disease with immunotherapy against the ß-amyloid (Aß) peptide or with enzyme inhibitors to reduce Aß production have not yet resulted in effective treatment, suggesting that alternative strategies may be useful. Here we explore the possibility of targeting the toxicity associated with Aß aggregation by using the recombinant human (rh) Bri2 BRICHOS chaperone domain, mutated to act selectively against Aß42 oligomer generation and neurotoxicity in vitro. We find that treatment of Aß precursor protein (App) knockin mice with repeated intravenous injections of rh Bri2 BRICHOS R221E, from an age close to the start of development of Alzheimer's disease-like pathology, improves recognition and working memory, as assessed using novel object recognition and Y maze tests, and reduces Aß plaque deposition and activation of astrocytes and microglia. When treatment was started about 4 months after Alzheimer's disease-like pathology was already established, memory improvement was not detected, but Aß plaque deposition and gliosis were reduced, and substantially reduced astrocyte accumulation in the vicinity of Aß plaques was observed. The degrees of treatment effects observed in the App knockin mouse models apparently correlate with the amounts of Bri2 BRICHOS detected in brain sections after the end of the treatment period.

Differential expression of striatal proteins in a mouse model of DOPA-responsive dystonia reveals shared mechanisms among dystonic disorders.

Dystonia is characterized by involuntary muscle contractions that cause debilitating twisting movements and postures. Although dysfunction of the basal ganglia, a brain region that mediates movement, is implicated in many forms of dystonia, the underlying mechanisms are unclear. The inherited metabolic disorder DOPA-responsive dystonia is considered a prototype for understanding basal ganglia dysfunction in dystonia because it is caused by mutations in genes necessary for the synthesis of the neurotransmitter dopamine, which mediates the activity of the basal ganglia. Therefore, to reveal abnormal striatal cellular processes and pathways implicated in dystonia, we used an unbiased proteomic approach in a knockin mouse model of DOPA-responsive dystonia, a model in which the striatum is known to play a central role in the expression of dystonia. Fifty-seven of the 1805 proteins identified were differentially regulated in DOPA-responsive dystonia mice compared to control mice. Most differentially regulated proteins were associated with gene ontology terms that implicated either mitochondrial or synaptic dysfunction whereby proteins associated with mitochondrial function were generally over-represented and proteins associated with synaptic function were largely under-represented. Remarkably, nearly 20% of the differentially regulated striatal proteins identified in our screen are associated with pathogenic variants that cause inherited disorders with dystonia as a sign in humans suggesting shared mechanisms across many different forms of dystonia.

Generation and pathological characterization of a transgenic mouse model carrying a missense PJVK mutation.

Hearing loss is the most prevalent hereditary sensory disorder in children. Approximately 2 in 1000 infants are affected by genetic hearing loss. The PJVK gene, which encodes the pejvakin protein, has been linked to autosomal recessive non-syndromic hearing loss DFNB59. Previous clinical studies have revealed that PJVK mutations might be associated with a wide spectrum of auditory manifestations, ranging from hearing loss of pure cochlear origin to that involving the retrocochlear central auditory pathway. The phenotypic variety makes the pathogenesis of this disease difficult to determine. Similarly, mouse models carrying different Pjvk defects show phenotypic variability and inconsistency. In this study, we generated a knockin mouse model carrying the c.874G > A (p.G292R) variant to model and investigate the auditory and vestibular phenotypes of DFNB59.

Nigrostriatal pathology with reduced astrocytes in LRRK2 S910/S935 phosphorylation deficient knockin mice.

Leucine-rich repeat kinase 2 (LRRK2) is genetically implicated in both familial and sporadic Parkinson's disease (PD). Moreover, LRRK2 has emerged as a compelling therapeutic target for the treatment of PD. Consequently, there is much interest in understanding LRRK2 and its role in PD pathogenesis. LRRK2 is constitutively phosphorylated on two serines, S910 and S935, that are required for interaction of LRRK2 with members of the 14-3-3 family of scaffolding proteins. Pathogenic LRRK2 missense mutations impair the phosphorylation of LRRK2 at these sites, but whether this contributes to PD pathology is unclear. To better understand how loss of LRRK2 phosphorylation relates to PD pathology, we have studied double knockin mice in which Lrrk2's serine 910 and 935 have both been mutated to alanine and can therefore no longer be phosphorylated. Nigrostriatal PD pathology was assessed in adult mice, aged mice, and mice inoculated with α-synuclein fibrils. Under all paradigms there was evidence of early PD pathology in the striatum of the knockin mice, namely alterations in dopamine regulating proteins and accumulation of α-synuclein. Striatal pathology was accompanied by a significant decrease in the number of astrocytes in the knockin mice. Despite striatal pathology, there was no degeneration of dopamine neurons in the substantia nigra and no evidence of a PD motor phenotype in the knockin mice. Our results suggest that modulation of LRRK2 serine 910 and 935 phosphorylation sites may have implications for dopamine turnover and astrocyte function, but loss of phosphorylation at these residues is not sufficient to induce PD neurodegeneration.

Absence of the dermatan sulfate chain of decorin does not affect mouse development.

BACKGROUND: In vitro studies suggest that the multiple functions of decorin are related to both its core protein and its dermatan sulfate chain. To determine the contribution of the dermatan sulfate chain to the functional properties of decorin in vivo, a mutant mouse whose decorin lacked a dermatan sulfate chain was generated. RESULTS: Homozygous mice expressing only the decorin core protein developed and grew in a similar manner to wild type mice. In both embryonic and postnatal mice, all connective tissues studied, including cartilage, skin and cornea, appeared to be normal upon histological examination, and their collagen fibrils were of normal diameter and organization. In addition, abdominal skin wounds healed in an identical manner in the mutant and wild type mice. CONCLUSIONS: The absence of a dermatan sulfate chain on decorin does not appear to overtly influence its functional properties in vivo.

Mechanism underlying unaltered cortical inhibitory synaptic transmission in contrast with enhanced excitatory transmission in CaV2.1 knockin migraine mice.

Familial hemiplegic migraine type 1 (FHM1), a monogenic subtype of migraine with aura, is caused by gain-of-function mutations in CaV2.1 (P/Q-type) calcium channels. In FHM1 knockin mice, excitatory neurotransmission at cortical pyramidal cell synapses is enhanced, but inhibitory neurotransmission at connected pairs of fast-spiking (FS) interneurons and pyramidal cells is unaltered, despite being initiated by CaV2.1 channels. The mechanism underlying the unaltered GABA release at cortical FS interneuron synapses remains unknown. Here, we show that the FHM1 R192Q mutation does not affect inhibitory transmission at autapses of cortical FS and other types of multipolar interneurons in microculture from R192Q knockin mice, and investigate the underlying mechanism. Lowering the extracellular [Ca(2+)] did not reveal gain-of-function of evoked transmission neither in control nor after prolongation of the action potential (AP) with tetraethylammonium, indicating unaltered AP-evoked presynaptic calcium influx at inhibitory autapses in FHM1 KI mice. Neither saturation of the presynaptic calcium sensor nor short duration of the AP can explain the unaltered inhibitory transmission in the mutant mice. Recordings of the P/Q-type calcium current in multipolar interneurons in microculture revealed that the current density and the gating properties of the CaV2.1 channels expressed in these interneurons are barely affected by the FHM1 mutation, in contrast with the enhanced current density and left-shifted activation gating of mutant CaV2.1 channels in cortical pyramidal cells. Our findings suggest that expression of specific CaV2.1 channels differentially sensitive to modulation by FHM1 mutations in inhibitory and excitatory cortical neurons underlies the gain-of-function of excitatory but unaltered inhibitory synaptic transmission and the likely consequent dysregulation of the cortical excitatory-inhibitory balance in FHM1.

A fluorescent splice-switching mouse model enables high-throughput, sensitive quantification of antisense oligonucleotide delivery and activity.

While antisense oligonucleotides (ASOs) are used in the clinic, therapeutic development is hindered by the inability to assay ASO delivery and activity in vivo. Accordingly, we developed a dual-fluorescence, knockin mouse model that constitutively expresses mKate2 and an engineered EGFP that is alternatively spliced in the presence of ASO to induce expression. We first examined free ASO activity in the brain following intracerebroventricular injection revealing EGFP splice-switching is both ASO concentration and time dependent in major central nervous system cell types. We then assayed the impact of lipid nanoparticle delivery on ASO activity after intravenous administration. Robust EGFP fluorescence was observed in the liver and EGFP+ cells were successfully isolated using fluorescence-activated cell sorting. Together, these results show the utility of this animal model in quantifying both cell-type- and organ-specific ASO delivery, which can be used to advance ASO therapeutics for many disease indications.

The µ-opioid receptor agonist DAMGO presynaptically suppresses solitary tract-evoked input to neurons in the rostral solitary nucleus.

Taste processing in the rostral nucleus of the solitary tract (rNST) is subject to modulatory influences including opioid peptides. Behavioral pharmacological studies suggest an influence of µ-opioid receptors in rNST, but the underlying mechanism is unknown. To determine the cellular site of action, we tested the effects of the µ-opioid receptor agonist DAMGO in vitro. Whole cell patch-clamp recordings were made in brain stem slices from GAD67-GFP knockin mice expressing enhanced green fluorescent protein (EGFP) under the control of the endogenous promoter for GAD67, a synthetic enzyme for GABA. Neuron counts showed that ∼36% of rNST neurons express GABA. We recorded monosynaptic solitary tract (ST)-evoked currents (jitter ≤ 300 µs) in both GAD67-EGFP-positive (GAD67+) and GAD67-EGFP-negative (GAD67-) neurons with equal frequency (25/31; 22/28), but the inputs to the GAD67+ neurons had significantly smaller paired-pulse ratios compared with GAD67- neurons. DAMGO (0.3 µM) significantly suppressed ST-evoked currents in both cell types (mean suppression = 46 ± 3.3% SE), significantly increased the paired-pulse ratio of these currents, and reduced the frequency of spontaneous miniature excitatory postsynaptic currents but did not diminish their amplitude, indicating a presynaptic site of action. Under inhibitory amino acid receptor blockade, DAMGO was significantly more suppressive in GAD67+ neurons (59% reduction) compared with GAD67- neurons (35% reduction), while the reverse was true in normal artificial cerebrospinal fluid (GAD67+: 35% reduction; GAD67-: 57% reduction). These findings suggest that DAMGO suppresses activity in rNST neurons predominantly via a presynaptic mechanism, and that this effect may interact significantly with tonic or evoked inhibitory activity.

Efficient CRISPR/Cas9-Assisted Knockin of Large DNA Donors by Pronuclear Microinjection During S-Phase in Mouse Zygotes.

In the CRISPR/Cas9-mediated gene cassette knockin (KI) strategy, a gene cassette is integrated into a target locus through a proper DNA repair pathway after the Cas9-induced double-strand DNA breaks; the activation of the DNA repair pathway is known to be correlated with the cell cycle. Recently, we have reported a new KI approach named SPRINT (S-phase pronuclear injection for targeting)-CRISPR, focusing on the correlation between the cell cycle and the KI efficiency in the mouse zygote microinjection. Our results suggest that the CRISPR-mediated KI with a homologous recombination-based donor vector during S-phase enhances the KI efficiency. For SPRINT-CRISPR, the uniformity of the zygotes in the cell cycle is achieved by in vitro fertilization, and the zygotes are cryopreserved until use. These reproductive techniques are necessary for efficient KI. Furthermore, Piezo-assisted microinjection has been successful in improving the survival rate of the injected embryos. In this chapter, we describe the protocols that focus on the zygote preparation and Piezo-assisted microinjection of the SPRINT-CRISPR method.

Generation and Application of Inducible Chimeric RNA ASTN2-PAPPAas Knockin Mouse Model.

Chimeric RNAs (chiRNAs) play many previously unrecognized roles in different diseases including cancer. They can not only be used as biomarkers for diagnosis and prognosis of various diseases but also serve as potential therapeutic targets. In order to better understand the roles of chiRNAs in pathogenesis, we inserted human sequences into mouse genome and established a knockin mouse model of the tamoxifen-inducible expression of ASTN2-PAPPA antisense chimeric RNA (A-PaschiRNA). Mice carrying the A-PaschiRNA knockin gene do not display any apparent abnormalities in growth, fertility, histological, hematopoietic, and biochemical indices. Using this model, we dissected the role of A-PaschiRNA in chemical carcinogen 4-nitroquinoline 1-oxide (4NQO)-induced carcinogenesis of esophageal squamous cell carcinoma (ESCC). To our knowledge, we are the first to generate a chiRNA knockin mouse model using the Cre-loxP system. The model could be used to explore the roles of chiRNA in pathogenesis and potential targeted therapies.

Plaque contact and unimpaired Trem2 is required for the microglial response to amyloid pathology.

Using spatial cell-type-enriched transcriptomics, we compare plaque-induced gene (PIG) expression in microglia-touching plaques, neighboring plaques, and far from plaques in an aged Alzheimer's mouse model with late plaque development. In 18-month-old APPNL-F/NL-F knockin mice, with and without the Alzheimer's disease risk mutation Trem2R47H/R47H, we report that expression of 38/55 PIGs have plaque-induced microglial upregulation, with a subset only upregulating in microglia directly contacting plaques. For seven PIGs, including Trem2, this upregulation is prevented in APPNL-F/NL-FTrem2R47H/R47H mice. These TREM2-dependent genes are all involved in phagocytic and degradative processes that we show correspond to a decrease in phagocytic markers and an increase in the density of small plaques in Trem2-mutated mice. Furthermore, despite the R47H mutation preventing increased Trem2 gene expression, TREM2 protein levels and microglial density are still marginally increased on plaques. Hence, both microglial contact with plaques and functioning TREM2 are necessary for microglia to respond appropriately to amyloid pathology.

Functional recovery of a novel knockin mouse model of dysferlinopathy by readthrough of nonsense mutation.

Biallelic mutations in the dysferlin gene cause limb-girdle muscular dystrophy 2B or Miyoshi distal myopathy. We found that nonsense mutations are the most common mutation type among Korean patients with dysferlinopathy; more than half of the patients have at least one nonsense allele, which may be amenable to readthrough therapy. We generated a knockin mouse, dqx, harboring DYSF p.Q832∗ mutation. Homozygous dqx mice lacked dysferlin in skeletal muscle, while 2 weeks of oral ataluren restored dysferlin expression and ameliorated skeletal muscle pathology. Their physical performance improved, and protection against eccentric contractions was noted. The improvement was most evident in mice treated with oral ataluren of 0.9 mg/mL. These improvements were sustained for 8 weeks in ataluren-treated dqx mice, while the parameters of A/J mice treated with ataluren over the same period did not improve. These results support that readthrough therapy by oral ataluren may also be applicable to dysferlinopathy patients with nonsense mutation.

Pronuclear Microinjection during S-Phase Increases the Efficiency of CRISPR-Cas9-Assisted Knockin of Large DNA Donors in Mouse Zygotes.

In CRISPR-Cas9-assisted knockin (KI) in zygotes, a remaining challenge is routinely achieving high-efficiency KI of large (kilobase-sized) DNA elements. Here, we focus on the timing of pronuclear injection and establish a reliable homologous recombination (HR)-based method to generate large KIs in zygotes compared with two other types of KI strategies involving distinct DNA repair pathways. At the ROSA26 locus, pronuclear injection with CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and Cas9 protein at the S phase by using the HR-based method yields the most efficient and accurate KIs (up to 70%). This approach is also generally effective for generating large KI alleles at other gene loci. We further apply our method to efficiently obtain biallelic ROSA26 KIs by sequential injection into both pronuclei. Our results suggest that delivery of genome editing components and donor DNA into S-phase zygotes is critical for efficient KI of large DNA elements.

Age-dependent accumulation of oligomeric SNCA/α-synuclein from impaired degradation in mutant LRRK2 knockin mouse model of Parkinson disease: role for therapeutic activation of chaperone-mediated autophagy (CMA).

Parkinson disease (PD) is an age-related neurodegenerative disorder associated with misfolded SNCA/α-synuclein accumulation in brain. Impaired catabolism of SNCA potentiates formation of its toxic oligomers. LRRK2 (leucine-rich repeat kinase-2) mutations predispose to familial and sporadic PD. Mutant LRRK2 perturbs chaperone-mediated-autophagy (CMA) to degrade SNCA. We showed greater age-dependent accumulation of oligomeric SNCA in striatum and cortex of aged LRRK2R1441G knockin (KI) mice, compared to age-matched wildtype (WT) by 53% and 31%, respectively. Lysosomal clustering and accumulation of CMA-specific LAMP2A and HSPA8/HSC70 proteins were observed in aged mutant striatum along with increased GAPDH (CMA substrate) by immunohistochemistry of dorsal striatum and flow cytometry of ventral midbrain cells. Using our new reporter protein clearance assay, mutant mouse embryonic fibroblasts (MEFs) expressing either SNCA or CMA recognition 'KFERQ'-like motif conjugated with photoactivated-PAmCherry showed slower cellular clearance compared to WT by 28% and 34%, respectively. However, such difference was not observed after the 'KFERQ'-motif was mutated. LRRK2 mutant MEFs exhibited lower lysosomal degradation than WT indicating lysosomal dysfunction. LAMP2A-knockdown reduced total lysosomal activity and clearance of 'KFERQ'-substrate in WT but not in mutant MEFs, indicating impaired CMA in the latter. A CMA-specific activator, AR7, induced neuronal LAMP2A transcription and lysosomal activity in MEFs. AR7 also attenuated the progressive accumulation of both intracellular and extracellular SNCA oligomers in prolonged cultures of mutant cortical neurons (DIV21), indicating that oligomer accumulation can be suppressed by CMA activation. Activation of autophagic pathways to reduce aged-related accumulation of pathogenic SNCA oligomers is a viable disease-modifying therapeutic strategy for PD.Abbreviations: 3-MA: 3-methyladenine; AR7: 7-chloro-3-(4-methylphenyl)-2H-1,4-benzoxazine; CMA: chaperone-mediated autophagy; CQ: chloroquine; CSF: cerebrospinal fluid; DDM: n-dodecyl ß-D-maltoside; DIV: days in vitro; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GWAS: genome-wide association studies; HSPA8/HSC70: heat shock protein 8; KFERQ: CMA recognition pentapeptide; KI: knockin; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; LDH: lactate dehydrogenase; LRRK2: leucine-rich repeat kinase 2; MEF: mouse embryonic fibroblast; NDUFS4: NADH:ubiquinone oxidoreductase core subunit S4; NE: novel epitope; PD: Parkinson disease; RARA/RARα: retinoic acid receptor, alpha; SNCA: synuclein, alpha; TUBB3/TUJ1: tubulin, beta 3 class III; WT: wild-type.

A missense point mutation in nerve growth factor (NGFR100W) results in selective peripheral sensory neuropathy.

The R100W mutation in nerve growth factor is associated with hereditary sensory autonomic neuropathy V in a Swedish family. These patients develop severe loss of perception to deep pain but with apparently normal cognitive functions. To better understand the disease mechanism, we examined a knockin mouse model of HSAN V. The homozygous mice showed significant structural deficits in intra-epidermal nerve fibers (IENFs) at birth. These mice had a total loss of pain perception at ∼2 months of age and often failed to survive to adulthood. Heterozygous mutant mice developed a progressive degeneration of small sensory fibers both behaviorally and functionally: they showed a progressive loss of IENFs starting at the age of 9 months accompanied with progressive loss of perception to painful stimuli such as noxious temperature. Quantitative analysis of lumbar 4/5 dorsal root ganglia revealed a significant reduction in small size neurons, while analysis of sciatic nerve fibers revealed the heterozygous mutant mice had no reduction in myelinated nerve fibers. Significantly, the amount of NGF secreted from mouse embryonic fibroblasts were reduced from both heterozygous and homozygous mice compared to their wild-type littermates. Interestingly, the heterozygous mice showed no apparent structural alteration in the brain: neither the anterior cingulate cortex nor the medial septum including NGF-dependent basal forebrain cholinergic neurons. Accordingly, these animals did not develop appreciable deficits in tests for brain function. Our study has thus demonstrated that the NGFR100W mutation likely affects the structure and function of peripheral sensory neurons.

Mutant TDP-43 Causes Early-Stage Dose-Dependent Motor Neuron Degeneration in a TARDBP Knockin Mouse Model of ALS.

Rare mutations in TARDBP, the gene encoding TDP-43, cause amyotrophic lateral sclerosis (ALS), and TDP-43 pathology is seen in a large majority of ALS patients, suggesting a central pathogenic role of this regulatory protein. The consequences of TARDBP mutations on TDP-43 function and the mechanism by which mutant TDP-43 causes neurodegeneration remain uncertain. Here, we characterize a series of knockin mice carrying disease-associated TARDBP mutations. We demonstrate that TDP-43M337V and TDP-43G298S are functional, each rescuing the lethality of TDP-43 loss of function. In a subset of aged heterozygous knockin mice, we observe the earliest signs of selective motor neuron degeneration, demonstrating that physiological levels of mutant TDP-43 are sufficient to initiate disease. Furthermore, aged homozygous mutants develop selective, asymmetric motor neuron pathology, providing in vivo evidence of TDP-43 dose-dependent neurotoxicity. These knockin mice represent a faithful in vivo model of early-stage ALS and enable future exploration of TDP-43-associated neurodegeneration.

Genome Editing in Mice Using CRISPR/Cas9 Technology.

CRISPR/Cas9 technology has revolutionized genome editing in mice, allowing for simple and rapid development of knockouts and knockins. CRISPR relies on small guide RNAs that direct the RNA-guided nuclease Cas9 to a designated genomic site using ∼20 bp of corresponding sequence. Cas9 then creates a double-strand break in the targeted loci that is either patched in an error-prone fashion to produce a frame-shift mutation, a knockout, or is repaired by recombination with donor DNA containing homology arms, a knockin. This protocol covers the techniques needed to rapidly generate knockout and knockin mice with CRISPR via microinjection of Cas9, the guide RNA, and possible donor DNA into the mouse zygote. © 2018 by John Wiley & Sons, Inc.

Mutation-induced loss of APP function causes GABAergic depletion in recessive familial Alzheimer's disease: analysis of Osaka mutation-knockin mice.

The E693Δ (Osaka) mutation in APP is linked to familial Alzheimer's disease. While this mutation accelerates amyloid ß (Aß) oligomerization, only patient homozygotes suffer from dementia, implying that this mutation is recessive and causes loss-of-function of amyloid precursor protein (APP). To investigate the recessive trait, we generated a new mouse model by knocking-in the Osaka mutation into endogenous mouse APP. The produced homozygous, heterozygous, and non-knockin littermates were compared for memory, neuropathology, and synaptic plasticity. Homozygotes showed memory impairment at 4 months, whereas heterozygotes did not, even at 8 months. Immunohistochemical and biochemical analyses revealed that only homozygotes displayed intraneuronal accumulation of Aß oligomers at 8 months, followed by abnormal tau phosphorylation, synapse loss, glial activation, and neuron loss. These pathologies were not observed at younger ages, suggesting that a certain mechanism other than Aß accumulation underlies the memory disturbance at 4 months. For the electrophysiology studies at 4 months, high-frequency stimulation evoked long-term potentiation in all mice in the presence of picrotoxin, but in the absence of picrotoxin, such potentiation was observed only in homozygotes, suggesting their GABAergic deficit. In support of this, the levels of GABA-related proteins and the number of dentate GABAergic interneurons were decreased in 4-month-old homozygotes. Since APP has been shown to play a role in dentate GABAergic synapse formation, the observed GABAergic depletion is likely associated with an impairment of the APP function presumably caused by the Osaka mutation. Oral administration of diazepam to homozygotes from 6 months improved memory at 8 months, and furthermore, prevented Aß oligomer accumulation, indicating that GABAergic deficiency is a cause of memory impairment and also a driving force of Aß accumulation. Our findings suggest that the Osaka mutation causes loss of APP function, leading to GABAergic depletion and memory disorder when wild-type APP is absent, providing a mechanism of the recessive heredity.

Chd8 Mutation Leads to Autistic-like Behaviors and Impaired Striatal Circuits.

Autism spectrum disorder (ASD) is a heterogeneous disease, but genetically defined models can provide an entry point to studying the molecular underpinnings of this disorder. We generated germline mutant mice with loss-of-function mutations in Chd8, a de novo mutation strongly associated with ASD, and demonstrate that these mice display hallmark ASD behaviors, macrocephaly, and craniofacial abnormalities similar to patient phenotypes. Chd8+/- mice display a broad, brain-region-specific dysregulation of major regulatory and cellular processes, most notably histone and chromatin modification, mRNA and protein processing, Wnt signaling, and cell-cycle regulation. We also find altered synaptic physiology in medium spiny neurons of the nucleus accumbens. Perturbation of Chd8 in adult mice recapitulates improved acquired motor learning behavior found in Chd8+/- animals, suggesting a role for CHD8 in adult striatal circuits. These results support a mechanism linking chromatin modification to striatal dysfunction and the molecular pathology of ASD.