α-Actinin-4-Mediated FSGS: An Inherited Kidney Disease Caused by an Aggregated and Rapidly Degraded Cytoskeletal Protein

Focal segmental glomerulosclerosis (FSGS) is a common pattern of renal injury, seen as both a primary disorder and as a consequence of underlying insults such as diabetes, HIV infection, and hypertension. Point mutations in theα-actinin-4 gene ACTN4 cause an autosomal dominant form of human FSGS. We characterized the biological effect of these mutations by biochemical assays, cell-based studies, and the development of a new mouse model. We found that a fraction of the mutant protein forms large aggregates with a high sedimentation coefficient. Localization of mutant α-actinin-4 in transfected and injected cells, as well as in situ glomeruli, showed aggregates of the mutant protein. Video microscopy showed the mutant α-actinin-4 to be markedly less dynamic than the wild-type protein. We developed a “knockin” mouse model by replacing Actn4 with a copy of the gene bearing an FSGS-associated point mutation. We used cells from these mice to show increased degradation of mutant α-actinin-4, mediated, at least in part, by the ubiquitin–proteasome pathway. We correlate these findings with studies of α-actinin-4 expression in human samples. “Knockin” mice with a disease-associated Actn4 mutation develop a phenotype similar to that observed in humans. Comparison of the phenotype in wild-type, heterozygous, and homozygous Actn4 “knockin” and “knockout” mice, together with our in vitro data, suggests that the phenotypes in mice and humans involve both gain-of-function and loss-of-function mechanisms.     

α-Actinin-4-Mediated FSGS: An Inherited Kidney Disease Caused by an Aggregated and Rapidly Degraded Cytoskeletal Protein

Focal segmental glomerulosclerosis (FSGS) is a common pattern of renal injury, seen as both a primary disorder and as a consequence of underlying insults such as diabetes, HIV infection, and hypertension. Point mutations in theα-actinin-4 gene ACTN4 cause an autosomal dominant form of human FSGS. We characterized the biological effect of these mutations by biochemical assays, cell-based studies, and the development of a new mouse model. We found that a fraction of the mutant protein forms large aggregates with a high sedimentation coefficient. Localization of mutant α-actinin-4 in transfected and injected cells, as well as in situ glomeruli, showed aggregates of the mutant protein. Video microscopy showed the mutant α-actinin-4 to be markedly less dynamic than the wild-type protein. We developed a “knockin” mouse model by replacing Actn4 with a copy of the gene bearing an FSGS-associated point mutation. We used cells from these mice to show increased degradation of mutant α-actinin-4, mediated, at least in part, by the ubiquitin–proteasome pathway. We correlate these findings with studies of α-actinin-4 expression in human samples. “Knockin” mice with a disease-associated Actn4 mutation develop a phenotype similar to that observed in humans. Comparison of the phenotype in wild-type, heterozygous, and homozygous Actn4 “knockin” and “knockout” mice, together with our in vitro data, suggests that the phenotypes in mice and humans involve both gain-of-function and loss-of-function mechanisms.     

Nonlinear Viscoelasticity of Actin Transiently Cross-linked with Mutant α-Actinin-4

Filamentous actin and associated actin binding proteins play an essential role in governing the mechanical properties of eukaryotic cells. They can also play a critical role in disease; for example, mutations in α-actinin-4 (Actn4), a dynamic actin cross-linking protein, cause proteinuric disease in humans and mice. Amino acid substitutions strongly affect the binding affinity and protein structure of Actn4. To study the physical impact of such substitutions on the underlying cytoskeletal network, we examine the bulk mechanical behavior of in vitro actin networks cross-linked with wild-type and mutant Actn4. These networks exhibit a complex viscoelastic response and are characterized by fluid-like behavior at the longest timescales, a feature that can be quantitatively accounted for through a model governed by dynamic cross-linking. The elastic behavior of the network is highly nonlinear, becoming much stiffer with applied stress. This nonlinear elastic response is also highly sensitive to the mutations of Actn4. In particular, we observe that actin networks cross-linked with Actn4 bearing the disease-causing K255E mutation are more brittle, with a lower breaking stress in comparison to networks cross-linked with wild-type Actn4. Furthermore, a mutation that ablates the first actin binding site (ABS1) in Actn4 abrogates the network's ability to stress-stiffen is standard nomenclature. These changes in the mechanical properties of actin networks cross-linked with mutant Actn4 may represent physical determinants of the underlying disease mechanism in inherited focal segmental glomerulosclerosis.     

Mice carrying a R142C Notch 3 knock-in mutation do not develop a CADASIL-like phenotype

CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy, MIM 125310) is a genetic vascular dementia disease that is linked to missense mutations, small in-frame deletions, and splice site mutations in the human Notch 3 gene. Here we describe the generation of a mouse knockin model for one of the most prevalent CADASIL mutations, an arginine to cysteine transition at position 141, R141C, which corresponds to mutation R142C in mouse NOTCH 3. CADASIL(R142C) mice show no apparent CADASIL-like phenotype after histological and MRI analysis. The NOTCH 3 (R142C) receptor is processed normally and does not appear to accumulate the ectodomain, which has been observed in CADASIL patients. We discuss possible reasons for the different outcomes of the same germline CADASIL mutation in mice and humans.     

Charcot-Marie-Tooth-linked mutant GARS is toxic to peripheral neurons independent of wild-type GARS levels

Charcot-Marie-Tooth disease type 2D (CMT2D) is a dominantly inherited peripheral neuropathy caused by missense mutations in the glycyl-tRNA synthetase gene (GARS). In addition to GARS, mutations in three other tRNA synthetase genes cause similar neuropathies, although the underlying mechanisms are not fully understood. To address this, we generated transgenic mice that ubiquitously over-express wild-type GARS and crossed them to two dominant mouse models of CMT2D to distinguish loss-of-function and gain-of-function mechanisms. Over-expression of wild-type GARS does not improve the neuropathy phenotype in heterozygous Gars mutant mice, as determined by histological, functional, and behavioral tests. Transgenic GARS is able to rescue a pathological point mutation as a homozygote or in complementation tests with a Gars null allele, demonstrating the functionality of the transgene and revealing a recessive loss-of-function component of the point mutation. Missense mutations as transgene-rescued homozygotes or compound heterozygotes have a more severe neuropathy than heterozygotes, indicating that increased dosage of the disease-causing alleles results in a more severe neurological phenotype, even in the presence of a wild-type transgene. We conclude that, although missense mutations of Gars may cause some loss of function, the dominant neuropathy phenotype observed in mice is caused by a dose-dependent gain of function that is not mitigated by over-expression of functional wild-type protein.     

Alpha-actinin-4 is required for normal podocyte adhesion

Mutations in the alpha-actinin-4 gene ACTN4 cause an autosomal dominant human kidney disease. Mice deficient in alpha-actinin-4 develop a recessive phenotype characterized by kidney failure, proteinuria, glomerulosclerosis, and retraction of glomerular podocyte foot processes. However, the mechanism by which alpha-actinin-4 deficiency leads to glomerular disease has not been defined. Here, we examined the effect of alpha-actinin-4 deficiency on the adhesive properties of podocytes in vivo and in a cell culture system. In alpha-actinin-4-deficient mice, we observed a decrease in the number of podocytes per glomerulus compared with wild-type mice as well as the presence of podocyte markers in the urine. Podocyte cell lines generated from alpha-actinin-4-deficient mice were less adherent than wild-type cells to glomerular basement membrane (GBM) components collagen IV and laminin 10 and 11. We also observed markedly reduced adhesion of alpha-actinin-4-deficient podocytes under increasing shear stresses. This adhesion deficit was restored by transfecting cells with alpha-actinin-4-GFP. We tested the strength of the integrin receptor-mediated linkages to the cytoskeleton by applying force to microbeads bound to integrin using magnetic pulling cytometry. Beads bound to alpha-actinin-4-deficient podocytes showed greater displacement in response to an applied force than those bound to wild-type cells. Consistent with integrin-dependent alpha-actinin-4-mediated adhesion, phosphorylation of beta1-integrins on alpha-actinin-4-deficient podocytes is reduced. We rescued the phosphorylation deficit by transfecting alpha-actinin-4 into alpha-actinin-4-deficient podocytes. These results suggest that alpha-actinin-4 interacts with integrins and strengthens the podocyte-GBM interaction thereby stabilizing glomerular architecture and preventing disease.     

A noncoding point mutation of Zeb1 causes multiple developmental malformations and obesity in Twirler mice

Heterozygous Twirler (Tw) mice develop obesity and circling behavior associated with malformations of the inner ear, whereas homozygous Tw mice have cleft palate and die shortly after birth. Zeb1 is a zinc finger protein that contributes to mesenchymal cell fate by repression of genes whose expression defines epithelial cell identity. This developmental pathway is disrupted in inner ears of Tw/Tw mice. The purpose of our study was to comprehensively characterize the Twirler phenotype and to identify the causative mutation. The Tw/+ inner ear phenotype includes irregularities of the semicircular canals, abnormal utricular otoconia, a shortened cochlear duct, and hearing loss, whereas Tw/Tw ears are severely malformed with barely recognizable anatomy. Tw/+ mice have obesity associated with insulin-resistance and have lymphoid organ hypoplasia. We identified a noncoding nucleotide substitution, c.58+181G>A, in the first intron of the Tw allele of Zeb1 (Zeb1(Tw)). A knockin mouse model of c.58+181G>A recapitulated the Tw phenotype, whereas a wild-type knockin control did not, confirming the mutation as pathogenic. c.58+181G>A does not affect splicing but disrupts a predicted site for Myb protein binding, which we confirmed in vitro. In comparison, homozygosity for a targeted deletion of exon 1 of mouse Zeb1, Zeb1(ΔEx1), is associated with a subtle abnormality of the lateral semicircular canal that is different than those in Tw mice. Expression analyses of E13.5 Twirler and Zeb1(ΔEx1) ears confirm that Zeb1(ΔEx1) is a null allele, whereas Zeb1(Tw) RNA is expressed at increased levels in comparison to wild-type Zeb1. We conclude that a noncoding point mutation of Zeb1 acts via a gain-of-function to disrupt regulation of Zeb1(Tw) expression, epithelial-mesenchymal cell fate or interactions, and structural development of the inner ear in Twirler mice. This is a novel mechanism underlying disorders of hearing or balance.     

A Novel TRPC6 Mutation That Causes Childhood FSGS

Background

TRPC6, encoding a member of the transient receptor potential (TRP) superfamily of ion channels, is a calcium-permeable cation channel, which mediates capacitive calcium entry into the cell. Until today, seven different mutations in TRPC6 have been identified as a cause of autosomal-dominant focal segmental glomerulosclerosis (FSGS) in adults.

Methodology/Principal Findings

Here we report a novel TRPC6 mutation that leads to early onset FSGS. We identified one family in whom disease segregated with a novel TRPC6 mutation (M132T), that also affected pediatric individuals as early as nine years of age. Twenty-one pedigrees compatible with an autosomal-dominant mode of inheritance and biopsy-proven FSGS were selected from a worldwide cohort of 550 families with steroid resistant nephrotic syndrome (SRNS). Whole cell current recordings of the mutant TRPC6 channel, compared to the wild-type channel, showed a 3 to 5-fold increase in the average out- and inward TRPC6 current amplitude. The mean inward calcium current of M132T was 10-fold larger than that of wild-type TRPC6. Interestingly, M132T mutants also lacked time-dependent inactivation. Generation of a novel double mutant M132T/N143S did not further augment TRPC6 channel activity.

Conclusions

In summary, our data shows that TRPC6 mediated FSGS can also be found in children. The large increase in channel currents and impaired channel inactivation caused by the M132T mutant leads to an aggressive phenotype that underlines the importance of calcium dose channeled through TRPC6.     

Elevated "Hydroxyl Radical" Generation In Vivo in an Animal Model of Amyotrophic Lateral Sclerosis

Abstract: Mutations in the enzyme copper/zinc superoxide dismutase-1 (SOD1) are associated with familial amyotrophic lateral sclerosis (FALS). The means by which the mutations cause FALS appears to be due to an adverse property of the mutant SOD1 protein that may involve increased generation of free radicals. We used in vivo microdialysis to measure the conversion of 4-hydroxybenzoic acid to 3,4-dihydroxybenzoic acid (3,4-DHBA) as a measure of "hydroxyl radical-like" production in transgenic amyotrophic lateral sclerosis (ALS) mice with the G93A mutation as well as littermate controls. The conversion of 4-hydroxybenzoic acid to 3,4-DHBA was significantly increased in the striatum of transgenic ALS mice at baseline but not in mice overexpressing wild-type human SOD1. Following administration of 3-nitropropionic acid 3,4-DHBA generation was significantly increased as compared with baseline, and the increase in the transgenic ALS mice was significantly greater than those in controls, whereas the increase in mice overexpressing wild-type human SOD1 was significantly attenuated. The present results provide in vivo evidence that expression of mutations in SOD1 can lead to increased generation of "hydroxyl radical-like" activity, which further implicates oxidative damage in the pathogenesis of ALS.     

The Molecular Basis of Brown, an Old Mouse Mutation, and of an Induced Revertant to Wild Type

The murine b locus encodes the tyrosinase related protein, TRP-1, a putative membrane-bound, copper-containing enzyme having about 40% amino acid identity with tyrosinase. The protein is essential for production of black rather than brown hair pigment. We show that skin of mutant brown mice contains the same amount of TRP-1 mRNA as wild type. On sequencing the coding region of the mutant mRNA we find four nucleotide differences from the wild-type (Black) sequence. Two of these differences result in different amino acid residues encoded by the brown allele. By sequencing the TRP-1 gene from a mouse in which a reversion from brown to Black has been induced by ethylnitrosourea we are able to show that only one of these amino acid changes, which substitutes a tyrosine for a conserved cysteine, is the cause of the brown phenotype. This mutation is adjacent to another cysteine at which, in the analogous position in tyrosinase a mutation results in the albino phenotype. The sequence of the revertant is the first report of DNA sequence of an ethylnitrosourea-induced genetic change in mouse.     

A humanized mouse model for a common beta0-thalassemia mutation

Accurate animal models that recapitulate the phenotype and genotype of patients with beta-thalassemia would enable the development of a range of possible therapeutic approaches. Here we report the generation of a mouse model carrying the codons 41-42 (-TTCT) beta-thalassemia mutation in the intact human beta-globin locus. This mutation accounts for approximately 40% of beta-thalassemia mutations in southern China and Thailand. We demonstrate a low level of production of gamma-globins from the mutant locus in day 18 embryos, as well as production of mutant human beta-globin mRNA. However, in contrast to transgenic mice carrying the normal human beta-globin locus, 4-bp deletion mice fail to show any phenotypic complementation of the knockout mutation of both murine beta-globin genes. Our studies suggest that this is a valuable model for gene correction in hemopoietic stem cells and for studying the effects of HbF inducers in vivo in a "humanized" thalassemic environment.     

Frequent recovery of triplet mutations in UVB-exposed skin epidermis of Xpc-knockout mice

Mutations of the Xpc gene cause a deficiency in global genome repair, a subpathway of nucleotide excision repair (NER), in mammalian cells. We used transgenic mice harboring the lambda-phage-based lacZ mutational reporter gene to study the effect of an Xpc null mutation (Xpc-/-) on damage induction, repair and mutagenesis in mouse skin epidermis after UVB irradiation. UVB induced equal amounts of cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (64PPs) in mouse skin epidermis of Xpc-/- and wild-type mice. CPDs were not significantly removed in either of the mouse genotypes by 12h after irradiation, whereas removal of 64PPs was observed in the wild-type. Irradiation with 300 and 400J/m2 UVB increased the lacZ mutant frequency in the Xpc-/- epidermis to at least twice as high as in the wild-type. Ninety-nine lacZ mutants isolated from the UVB-exposed epidermis of Xpc(-/-)mice were analyzed and compared with mutant sequences from irradiated wild-type mice. The spectra of the mutations in the two genotypes were both highly UV-specific and similar in the dominance of C-->T transitions at dipyrimidine sites; however, Xpc-/- mice had a higher frequency of two-base tandem substitutions, including CC-->TT mutations, three-base tandem substitutions and double base substitutions that were separated by one unchanged base in a three-base sequence (alternating mutations). These tandem/alternating mutations included a remarkably large number of triplet mutations, a recently reported, novel type of UV-specific mutation, characterized by multiple base substitutions or frameshifts within a three-nucleotide sequence containing a dipyrimidine. We concluded that the triplet mutation is a UV-specific mutation that preferably occurs in NER deficient genetic backgrounds.     

Haploinsufficiency for Translation Elongation Factor eEF1A2 in Aged Mouse Muscle and Neurons Is Compatible with Normal Function

Translation elongation factor isoform eEF1A2 is expressed in muscle and neurons. Deletion of eEF1A2 in mice gives rise to the neurodegenerative phenotype "wasted" (wst). Mice homozygous for the wasted mutation die of muscle wasting and neurodegeneration at four weeks post-natal. Although the mutation is said to be recessive, aged heterozygous mice have never been examined in detail; a number of other mouse models of motor neuron degeneration have recently been shown to have similar, albeit less severe, phenotypic abnormalities in the heterozygous state. We therefore examined the effects of ageing on a cohort of heterozygous +/wst mice and control mice, in order to establish whether a presumed 50% reduction in eEF1A2 expression was compatible with normal function. We evaluated the grip strength assay as a way of distinguishing between wasted and wild-type mice at 3-4 weeks, and then performed the same assay in older +/wst and wild-type mice. We also used rotarod performance and immunohistochemistry of spinal cord sections to evaluate the phenotype of aged heterozygous mice. Heterozygous mutant mice showed no deficit in neuromuscular function or signs of spinal cord pathology, in spite of the low levels of eEF1A2.     

Variable expression of Dkc1 mutations in mice

In humans mutations in DKC1, cause the rare bone marrow failure syndrome dyskeratosis congenita. We have used gene targeting to produce mouse ES cells with Dkc1 mutations that cause DC when in humans. The mutation A353V, the most common human mutation, causes typical DC to very severe DC in humans. Male chimeric mice carrying this mutation do not pass the mutated allele to their offspring. The mutation G402E accounts for a single typical case of DC in a human family. The allele carrying this mutation was transmitted to the offspring with high efficiency. Expression of RNA and protein was reduced compared to wild type animals, but no abnormalities of growth and development or in blood values were found in mutant mice. Thus Dkc1 mutations have variable expression inmice, as in humans. genesis 47:366–373, 2009. © 2009 Wiley‐Liss, Inc.     

The Clouston syndrome mutation connexin30 A88V leads to hyperproliferation of sebaceous glands and hearing impairments in mice

Distinct mutations in the gap junction protein connexin30 (Cx30) can cause the ectodermal dysplasia Clouston syndrome in humans. We have generated a new mouse line expressing the Clouston syndrome mutation Cx30A88V under the control of the endogenous Cx30 promoter. Our results show that the mutated Cx30A88V protein is incorporated in gap junctional plaques of the epidermis. Homozygous Cx30A88V mice reveal hyperproliferative and enlarged sebaceous glands as well as a mild palmoplantar hyperkeratosis. Additionally, homozygous mutant mice show an altered hearing profile compared to control mice. We conclude that the Cx30A88V mutation triggers hyperproliferation in the skin and changes the cochlear homeostasis in mice.     

Cataracts and Microphthalmia Caused by a Gja8 Mutation in Extracellular Loop 2

The mouse semi-dominant Nm2249 mutation displays variable cataracts in heterozygous mice and smaller lenses with severe cataracts in homozygous mice. This mutation is caused by a Gja8^R205G point mutation in the second extracellular loop of the Cx50 (or α8 connexin) protein. Immunohistological data reveal that Cx50-R205G mutant proteins and endogenous wild-type Cx46 (or α3 connexin) proteins form diffuse tiny spots rather than typical punctate signals of normal gap junctions in the lens. The level of phosphorylated Cx46 proteins is decreased in Gja8^R205G/R205G mutant lenses. Genetic analysis reveals that the Cx50-R205G mutation needs the presence of wild-type Cx46 to disrupt lens peripheral fibers and epithelial cells. Electrophysiological data in Xenopus oocytes reveal that Cx50-R205G mutant proteins block channel function of gap junctions composed of wild-type Cx50, but only affect the gating of wild-type Cx46 channels. Both genetic and electrophysiological results suggest that Cx50-R205G mutant proteins alone are unable to form functional channels. These findings imply that the Gja8^R205G mutation differentially impairs the functions of Cx50 and Cx46 to cause cataracts, small lenses and microphthalmia. The Gja8^R205G mutation occurs at the same conserved residue as the human GJA8^R198W mutation. This work provides molecular insights to understand the cataract and microphthalmia/microcornea phenotype caused by Gja8 mutations in mice and humans.     

Mice with podocyte-specific overexpression of wild type α-actinin-4 are healthy controls for K256E-α-actinin-4 mutant transgenic mice

Mutations in the gene ACTN4 encoding the actin bundling protein—α-actinin-4 underlie an inherited form of kidney lesions known as focal segmental glomerulosclerosis (FSGS). Previously, we developed a model for this condition by generating mice with podocyte-specific overexpression of a disease-causing mutant α-actinin-4 (K256E-ACTN4 ^pod+). However, whether α-actinin-4 overexpression artifacts and not the gain of affinity effects of the mutation accounted for the robust FSGS phenotype in these mice was unclear. To address this question, we developed a control line of mice with podocyte-specific overexpression of wildtype α-actinin-4 (wt-ACTN4 ^pod+). An 8.3 kb fragment of the mouse nephrin promoter (NPHS1) was used to drive expression of a hemagglutinin (HA)-tagged wildtype α-actinin-4 coding sequence in mice. Five founder lines expressing the HA-tagged α-actinin-4 protein in a podocyte-specific manner were obtained, as determined by co-immunofluorescence with HA and synaptopodin antibodies. Quantitative PCR revealed that renal transgene mRNA levels of wt-ACTN4 ^pod+ mice are similar to K256E-ACTN4 ^pod+ mice. In contrast to K256E-ACTN4 ^pod+ mice which exhibit albuminuria, podocyte foot process effacement and glomerular scarring, wt-ACTN4 ^pod+ mice are healthy and indistinguishable from non-transgenic littermates. These findings suggest that the K256E mutation itself and not overexpression of α-actinin-4 protein per se accounts for the FSGS phenotype in our transgenic model.     

Rational design of temperature-sensitive alleles using computational structure prediction

Temperature-sensitive (ts) mutations are mutations that exhibit a mutant phenotype at high or low temperatures and a wild-type phenotype at normal temperature. Temperature-sensitive mutants are valuable tools for geneticists, particularly in the study of essential genes. However, finding ts mutations typically relies on generating and screening many thousands of mutations, which is an expensive and labor-intensive process. Here we describe an in silico method that uses Rosetta and machine learning techniques to predict a highly accurate "top 5" list of ts mutations given the structure of a protein of interest. Rosetta is a protein structure prediction and design code, used here to model and score how proteins accommodate point mutations with side-chain and backbone movements. We show that integrating Rosetta relax-derived features with sequence-based features results in accurate temperature-sensitive mutation predictions.     

Cyclin-dependent kinase 4 hyperactivity promotes autoreactivity in the immune system but protects pancreatic cell mass from autoimmune destruction in the nonobese diabetic mouse model

Cyclin-dependent kinase 4 (Cdk4) plays a central role in perinatal pancreatic beta cell replication, thus becoming a potential target for therapeutics in autoimmune diabetes. Its hyperactive form, Cdk4R24C, causes beta cell hyperplasia without promoting hypoglycemia in a nonautoimmune-prone mouse strain. In this study, we explore whether beta cell hyperproliferation induced by the Cdk4R24C mutation balances the autoimmune attack against beta cells inherent to the NOD genetic background. To this end, we backcrossed the Cdk4R24C knockin mice, which have the Cdk4 gene replaced by the Cdk4R24C mutated form, onto the NOD genetic background. In this study, we show that NOD/Cdk4R24C knockin mice exhibit exacerbated diabetes and insulitis, and that this exacerbated diabetic phenotype is solely due to the hyperactivity of the NOD/Cdk4R24C immune repertoire. Thus, NOD/Cdk4R24C splenocytes confer exacerbated diabetes when adoptively transferred into NOD/SCID recipients, compared with NOD/wild-type (WT) donor splenocytes. Accordingly, NOD/Cdk4R24C splenocytes show increased basal proliferation and higher activation markers expression compared with NOD/WT splenocytes. However, to eliminate the effect of the Cdk4R24C mutation specifically in the lymphocyte compartment, we introduced this mutation into NOD/SCID mice. NOD/SCID/Cdk4R24C knockin mice develop beta cell hyperplasia spontaneously. Furthermore, NOD/SCID/Cdk4R24C knockin females that have been adoptively transferred with NOD/WT splenocytes are more resistant to autoimmunity than NOD/SCID WT female. Thus, the Cdk4R24C mutation opens two avenues in the NOD model: when expressed specifically in beta cells, it provides a new potential strategy for beta cell regeneration in autoimmune diabetes, but its expression in the immune repertoire exacerbates autoimmunity.