Type 1B diabetes (typically early onset; without islet autoantibodies) has been described in patients bearing small coding sequence mutations in the INS gene. Not all mutations in the INS gene cause the autosomal dominant Mutant INS-gene-induced Diabetes of Youth (MIDY) syndrome, but most missense mutations affecting proinsulin folding produce MIDY. MIDY patients are heterozygotes, with the expressed proinsulin mutants exerting dominant-negative (gain of toxic function) behavior in pancreatic beta cells. Herein, we focus primarily on proinsulin folding in the endoplasmic reticulum, providing insight into perturbations of this folding pathway in MIDY. Accumulated evidence indicates that in the molecular pathogenesis of the disease, misfolded proinsulin exerts dominant effects that initially inhibit insulin production, progressing to beta cell demise with diabetes.

The most common dominantly inherited ataxia, spinocerebellar ataxia type 3 (SCA3), is an incurable neurodegenerative disorder caused by a CAG repeat expansion in the ATXN3 gene that encodes an abnormally long polyglutamine tract in the disease protein, ATXN3. Mice lacking ATXN3 are phenotypically normal; hence, disease gene suppression offers a compelling approach to slow the neurodegenerative cascade in SCA3. Here we tested antisense oligonucleotides (ASOs) that target human ATXN3 in two complementary mouse models of SCA3: yeast artificial chromosome (YAC) MJD-Q84.2 (Q84) mice expressing the full-length human ATXN3 gene and cytomegalovirus (CMV) MJD-Q135 (Q135) mice expressing a human ATXN3 cDNA. Intracerebroventricular injection of ASOs resulted in widespread delivery to the most vulnerable brain regions in SCA3. In treated Q84 mice, three of five tested ASOs reduced disease protein levels by >50% in the diencephalon, cerebellum, and cervical spinal cord. Two ASOs also significantly reduced mutant ATXN3 in the mouse forebrain and resulted in no signs of astrogliosis or microgliosis. In Q135 mice expressing a single ATXN3 isoform via a cDNA transgene, ASOs did not result in similar robust ATXN3 silencing. Our results indicate that ASOs targeting full-length human ATXN3 would likely be well tolerated and could lead to a preventative therapy for SCA3.

It has previously been shown that misfolded mutant Akita proinsulin in the endoplasmic reticulum engages directly in protein complexes either with nonmutant proinsulin or with "hProCpepGFP" (human proinsulin bearing emerald-GFP within the C-peptide), impairing the trafficking of these "bystander" proinsulin molecules (Liu, M., Hodish, I., Rhodes, C. J., and Arvan, P. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 15841-15846). Herein, we generated transgenic mice, which, in addition to expressing endogenous proinsulin, exhibit ␤-cellspecific expression of hProCpepGFP via the Ins1 promoter. In these mice, hProCpepGFP protein levels are physiologically regulated, and hProCpepGFP is packaged and processed to CpepGFP that is co-stored in ␤-secretory granules. Visualization of CpepGFP fluorescence provides a quantifiable measure of pancreatic islet insulin content that can be followed in live animals in states of health and disease. We examined loss of pancreatic insulin in hProCpepGFP transgenic mice mated to Akita mice that develop neonatal diabetes because of the expression of misfolded proinsulin. Loss of bystander insulin in Akita animals is detected initially as a block in CpepGFP/insulin production with intracellular accumulation of the precursor, followed ultimately by loss of pancreatic ␤-cells. The data support that misfolded proinsulin perturbs bystander proinsulin in the endoplasmic reticulum, leading to ␤-cell failure.During the progression of diabetes mellitus, the endocrine pancreas encounters difficulty in meeting insulin requirements (1); ␤-cell dysfunction is recognized as a major contributor to the disease (2-5). One element of ␤-cell dysfunction is ER 3 stress (6 -11) with ER accumulation of misfolded protein (12), especially proinsulin (13, 14). ␤-Cells ordinarily maintain a high level of proinsulin production with finite additional capacity before the biosynthetic apparatus is taxed to the point of ER stress (15). Chronically increased secretory demand, either in animal models or in humans, results in morphological depletion of ␤-secretory granules with a compensatory increase in apparent secretory pathway activity, including distention of the ER (16 -18). These conditions may favor additional proinsulin misfolding (19).The causality between misfolded proinsulin and ␤-cell failure is unequivocally established in congenital diabetes caused by preproinsulin coding sequence mutations, in which diabetes is inherited in an autosomal dominant manner (20 -24). Insulin haploinsufficiency cannot account for the diabetes (25), yet despite three normal proinsulin alleles, both Akita and Munich mice each develop overt diabetes by expressing from a single allele a mutant proinsulin with replacement of one Cys residue that disrupts one of the three proinsulin disulfide bonds (26,27). In addition to being retained in the ER, it has been suggested that misfolded proinsulin may impair normal insulin production via physical interactions between mutant and wild-type proinsulin gene products (26). Indeed, we have direct...

Background:We have examined the effect of PDI knockdown in pancreatic ␤-cells. Results: Upon knockdown, proinsulin oxidation to form native disulfide bonds is enhanced and accompanied by improved exit from the ER with increased total insulin secretion. Conclusion:We hypothesize that PDI exhibits unfoldase activity for proinsulin. Significance: Unexpectedly, PDI increases retention of proinsulin within the ER of pancreatic ␤-cells.

NMDA receptors (NMDARs) play an essential role in some forms of synaptic plasticity, learning, and memory. Therefore, these receptors are highly regulated with respect to their localization, activation, and abundance both within and on the surface of mammalian neurons. Fundamental questions remain, however, regarding how this complex regulation is achieved. Using cell-based models and F-box Only Protein 2 (Fbxo2) knock-out mice, we found that the ubiquitin ligase substrate adaptor protein Fbxo2, previously reported to facilitate the degradation of the NMDAR subunit GluN1 in vitro, also functions to regulate GluN1 and GluN2A subunit levels in the adult mouse brain. In contrast, GluN2B subunit levels are not affected by the loss of Fbxo2. The loss of Fbxo2 results in greater surface localization of GluN1 and GluN2A, together with increases in the synaptic markers PSD-95 and Vglut1. These synaptic changes do not manifest as neurophysiological differences or alterations in dendritic spine density in Fbxo2 knock-out mice, but result instead in increased axo-dendritic shaft synapses. Together, these findings suggest that Fbxo2 controls the abundance and localization of specific NMDAR subunits in the brain and may influence synapse formation and maintenance.