MITF, a gene that is mutated in familial melanoma and Waardenburg syndrome, encodes multiple isoforms expressed from alternative promoters that share common coding exons but have unique amino termini. It is not completely understood how these isoforms influence pigmentation in different tissues and how the expression of these independent isoforms of MITF is regulated. Here, we show that melanocytes express two isoforms of MITF, MITF-A and MITF-M. The expression of MITF-A is partially regulated by a newly identified retinoid enhancer element located upstream of the MITF-A promoter. Mitf-A knockout mice have only subtle changes in melanin accumulation in the hair and reduced Tyr expression in the eye. In contrast, Mitf-M-null mice have enlarged kidneys, lack neural crest-derived melanocytes in the skin, choroid, and iris stroma, yet maintain pigmentation within the retinal pigment epithelium and iris pigment epithelium of the eye. Taken together, these studies identify a critical role for MITF-M in melanocytes, a minor role for MITF-A in regulating pigmentation in the hair and Tyr expression in the eye, and a novel role for MITF-M in size control of the kidney.

MITF, a gene that is mutated in familial melanoma and Waardenburg syndrome, encodes multiple isoforms expressed from alternative promoters that share common coding exons but have unique amino termini. It is not completely understood how these isoforms influence pigmentation in different tissues and how the expression of these independent isoforms of MITF is regulated. Here, we show that melanocytes express two isoforms of MITF, MITF-A and MITF-M. The expression of MITF-A is partially regulated by a newly identified retinoid enhancer element located upstream of the MITF-A promoter. Mitf-A knockout mice have only subtle changes in melanin accumulation in the hair and reduced Tyr expression in the eye. In contrast, Mitf-M-null mice have enlarged kidneys, lack neural crest-derived melanocytes in the skin, choroid, and iris stroma, yet maintain pigmentation within the retinal pigment epithelium and iris pigment epithelium of the eye. Taken together, these studies identify a critical role for MITF-M in melanocytes, a minor role for MITF-A in regulating pigmentation in the hair and Tyr expression in the eye, and a novel role for MITF-M in size control of the kidney.

Pompe disease, an autosomal recessive disorder caused by deficient lysosomal acid α-glucosidase (GAA), is characterized by accumulation of intra-lysosomal glycogen in skeletal and oftentimes cardiac muscle. The c.1935C>A (p.Asp645Glu) variant, the most frequent GAA pathogenic mutation in people of Southern Han Chinese ancestry, causes infantile-onset Pompe disease (IOPD), presenting neonatally with severe hypertrophic cardiomyopathy, profound muscle hypotonia, respiratory failure, and infantile mortality. We applied CRISPR-Cas9 homology-directed repair (HDR) using a novel dual sgRNA approach flanking the target site to generate a Gaa^em1935C>A knock-in mouse model and a myoblast cell line carrying the Gaa c.1935C>A mutation. Herein we describe the molecular, biochemical, histological, physiological, and behavioral characterization of 3-month-old homozygous Gaa^em1935C>A mice. Homozygous Gaa^em1935C>A knock-in mice exhibited normal Gaa mRNA expression levels relative to wild-type mice, had near-abolished GAA enzymatic activity, markedly increased tissue glycogen storage, and concomitantly impaired autophagy. Three-month-old mice demonstrated skeletal muscle weakness and hypertrophic cardiomyopathy but no premature mortality. The Gaa^em1935C>A knock-in mouse model recapitulates multiple salient aspects of human IOPD caused by the GAA c.1935C>A pathogenic variant. It is an ideal model to assess innovative therapies to treat IOPD, including personalized therapeutic strategies that correct pathogenic variants, restore GAA activity and produce functional phenotypes.

Background

Variants in the mitochondrial complex I assembly factor, NUBPL are associated with a rare cause of complex I deficiency mitochondrial disease. Patients affected by complex I deficiency harboring homozygous NUBPL variants typically have neurological problems including seizures, intellectual disability, and ataxia associated with cerebellar hypoplasia. Thus far only 19 cases have been reported worldwide, and no treatment is available for this rare disease.

Methods

To investigate the pathogenesis of NUBPL-associated complex I deficiency, and for translational studies, we generated a knock-in mouse harboring a patient-specific variant Nubpl c.311T>C; p. L104P reported in three families.

Results

Similar to Nubpl global knockout mice, the Nubpl p. L104P homozygous mice are lethal at embryonic day E10.5, suggesting that the Nubpl p. L104P variant is likely a hypomorph allele. Given the recent link between Parkinson's disease and loss-of-function NUBPL variants, we also explored aging-related behaviors and immunocytochemical changes in Nubpl hemizygous mice and did not find significant behavioral and pathological changes for alpha-synuclein and oxidative stress markers .

Conclusion

Our data suggest that homozygotes with Nubpl variants, similar to the null mice, are lethal, and heterozygotes are phenotypically and neuropathologically normal. We propose that a tissue-specific knockout strategy is required to establish a mouse model of Nubpl-associated complex I deficiency disorder for future mechanistic and translational studies.

Advances in genome sequencing have identified over 1300 mutations in the SCN1A sodium channel gene that result in genetic epilepsies. However, it still remains unclear how most individual mutations within SCN1A result in seizures. A previous study has shown that the K1270T (KT) mutation, linked to genetic epilepsy with febrile seizure plus (GEFS+) in humans, causes heat-induced seizure activity associated with a temperature-dependent decrease in GABAergic neuron excitability in a Drosophila knock-in model. To examine the behavioral and cellular effects of this mutation in mammals, we introduced the equivalent KT mutation into the mouse (Mus musculus) Scn1a (Scn1a^KT) gene using CRISPR/Cas9 and generated mutant lines in two widely used genetic backgrounds: C57BL/6NJ and 129X1/SvJ. In both backgrounds, mice homozygous for the KT mutation had spontaneous seizures and died by postnatal day (P)23. There was no difference in mortality of heterozygous KT mice compared with wild-type littermates up to six months old. Heterozygous mutants exhibited heat-induced seizures at ∼42°C, a temperature that did not induce seizures in wild-type littermates. In acute hippocampal slices at permissive temperatures, current-clamp recordings revealed a significantly depolarized shift in action potential threshold and reduced action potential amplitude in parvalbumin (PV)-expressing inhibitory CA1 interneurons in Scn1a^KT/+ mice. There was no change in the firing properties of excitatory CA1 pyramidal neurons. These results suggest that a constitutive decrease in inhibitory interneuron excitability contributes to the seizure phenotype in the mouse model.

Mouse models of human diseases are invaluable tools for studying pathogenic mechanisms and testing interventions and therapeutics. For disorders such as Alzheimer's disease in which numerous models are being generated, a challenging first step is to identify the most appropriate model and age to effectively evaluate new therapeutic approaches. Here we conducted a detailed phenotypic characterization of the 5xFAD model on a congenic C57BL/6 J strain background, across its lifespan - including a seldomly analyzed 18-month old time point to provide temporally correlated phenotyping of this model and a template for characterization of new models of LOAD as they are generated. This comprehensive analysis included quantification of plaque burden, Aβ biochemical levels, and neuropathology, neurophysiological measurements and behavioral and cognitive assessments, and evaluation of microglia, astrocytes, and neurons. Analysis of transcriptional changes was conducted using bulk-tissue generated RNA-seq data from microdissected cortices and hippocampi as a function of aging, which can be explored at the MODEL-AD Explorer and AD Knowledge Portal. This deep-phenotyping pipeline identified novel aspects of age-related pathology in the 5xFAD model.

Background

Variants in ABCA7, a member of the ABC transporter superfamily, have been associated with increased risk for developing late onset Alzheimer's disease (LOAD).

Methods

CRISPR-Cas9 was used to generate an Abca7^V1613M variant in mice, modeling the homologous human ABCA7^V1599M variant, and extensive characterization was performed.

Results

Abca7^V1613M microglia show differential gene expression profiles upon lipopolysaccharide challenge and increased phagocytic capacity. Homozygous Abca7^V1613M mice display elevated circulating cholesterol and altered brain lipid composition. When crossed with 5xFAD mice, homozygous Abca7^V1613M mice display fewer Thioflavin S-positive plaques, decreased amyloid beta (Aβ) peptides, and altered amyloid precursor protein processing and trafficking. They also exhibit reduced Aβ-associated inflammation, gliosis, and neuronal damage.

Discussion

Overall, homozygosity for the Abca7^V1613M variant influences phagocytosis, response to inflammation, lipid metabolism, Aβ pathology, and neuronal damage in mice. This variant may confer a gain of function and offer a protective effect against Alzheimer's disease-related pathology.

Highlights

ABCA7 recognized as a top 10 risk gene for developing Alzheimer's disease. Loss of function mutations result in increased risk for LOAD. V1613M variant reduces amyloid beta plaque burden in 5xFAD mice. V1613M variant modulates APP processing and trafficking in 5xFAD mice. V1613M variant reduces amyloid beta-associated damage in 5xFAD mice.

Animal models of disease are valuable resources for investigating pathogenic mechanisms and potential therapeutic interventions. However, for complex disorders such as Alzheimer's disease (AD), the generation and availability of innumerous distinct animal models present unique challenges to AD researchers and hinder the success of useful therapies. Here, we conducted an in-depth analysis of the 3xTg-AD mouse model of AD across its lifespan to better inform the field of the various pathologies that appear at specific ages, and comment on drift that has occurred in the development of pathology in this line since its development 20 years ago. This modern characterization of the 3xTg-AD model includes an assessment of impairments in long-term potentiation followed by quantification of amyloid beta (Aβ) plaque burden and neurofibrillary tau tangles, biochemical levels of Aβ and tau protein, and neuropathological markers such as gliosis and accumulation of dystrophic neurites. We also present a novel comparison of the 3xTg-AD model with the 5xFAD model using the same deep-phenotyping characterization pipeline and show plasma NfL is strongly driven by plaque burden. The results from these analyses are freely available via the AD Knowledge Portal (https://modeladexplorer.org/). Our work demonstrates the utility of a characterization pipeline that generates robust and standardized information relevant to investigating and comparing disease etiologies of current and future models of AD.

Background

The TREM2 R47H variant is one of the strongest genetic risk factors for late-onset Alzheimer's Disease (AD). Unfortunately, many current Trem2 ^R47H mouse models are associated with cryptic mRNA splicing of the mutant allele that produces a confounding reduction in protein product. To overcome this issue, we developed the Trem2^{R47H NSS} (Normal Splice Site) mouse model in which the Trem2 allele is expressed at a similar level to the wild-type Trem2 allele without evidence of cryptic splicing products.

Methods

Trem2^{R47H NSS} mice were treated with the demyelinating agent cuprizone, or crossed with the 5xFAD mouse model of amyloidosis, to explore the impact of the TREM2 R47H variant on inflammatory responses to demyelination, plaque development, and the brain's response to plaques.

Results

Trem2^{R47H NSS} mice display an appropriate inflammatory response to cuprizone challenge, and do not recapitulate the null allele in terms of impeded inflammatory responses to demyelination. Utilizing the 5xFAD mouse model, we report age- and disease-dependent changes in Trem2^{R47H NSS} mice in response to development of AD-like pathology. At an early (4-month-old) disease stage, hemizygous 5xFAD/homozygous Trem2^{R47H NSS} (5xFAD/Trem2^{R47H NSS}) mice have reduced size and number of microglia that display impaired interaction with plaques compared to microglia in age-matched 5xFAD hemizygous controls. This is associated with a suppressed inflammatory response but increased dystrophic neurites and axonal damage as measured by plasma neurofilament light chain (NfL) level. Homozygosity for Trem2^{R47H NSS} suppressed LTP deficits and loss of presynaptic puncta caused by the 5xFAD transgene array in 4-month-old mice. At a more advanced (12-month-old) disease stage 5xFAD/Trem2^{R47H NSS} mice no longer display impaired plaque-microglia interaction or suppressed inflammatory gene expression, although NfL levels remain elevated, and a unique interferon-related gene expression signature is seen. Twelve-month old Trem2^{R47H NSS} mice also display LTP deficits and postsynaptic loss.

Conclusions

The Trem2^{R47H NSS} mouse is a valuable model that can be used to investigate age-dependent effects of the AD-risk R47H mutation on TREM2 and microglial function including its effects on plaque development, microglial-plaque interaction, production of a unique interferon signature and associated tissue damage.

Introduction

The BIN1 coding variant rs138047593 (K358R) is linked to Late-Onset Alzheimer's Disease (LOAD) via targeted exome sequencing.

Methods

To elucidate the functional consequences of this rare coding variant on brain amyloidosis and neuroinflammation, we generated BIN1^K358R knock-in mice using CRISPR/Cas9 technology. These mice were subsequently bred with 5xFAD transgenic mice, which serve as a model for Alzheimer's pathology.

Results

The presence of the BIN1^K358R variant leads to increased cerebral amyloid deposition, with a dampened response of astrocytes and oligodendrocytes, but not microglia, at both the cellular and transcriptional levels. This correlates with decreased neurofilament light chain in both plasma and brain tissue. Synaptic densities are significantly increased in both wild-type and 5xFAD backgrounds homozygous for the BIN1^K358R variant.

Discussion

The BIN1 K358R variant modulates amyloid pathology in 5xFAD mice, attenuates the astrocytic and oligodendrocytic responses to amyloid plaques, decreases damage markers, and elevates synaptic densities.

Highlights

BIN1 rs138047593 (K358R) coding variant is associated with increased risk of LOAD. BIN1 K358R variant increases amyloid plaque load in 12-month-old 5xFAD mice. BIN1 K358R variant dampens astrocytic and oligodendrocytic response to plaques. BIN1 K358R variant decreases neuronal damage in 5xFAD mice. BIN1 K358R upregulates synaptic densities and modulates synaptic transmission.