Dimethyl fumarate ameliorates myoclonus stemming from protein misfolding in oligodendrocytes.

Multiple sclerosis (MS) is considered a primary autoimmune disease; however, this view is increasingly being challenged in basic and clinical science arenas because of the growing body of clinical trials' data showing that exclusion of immune cells from the CNS only modestly slows disease progression to disability. Accordingly, there is significant need for expanding the scope of potential disease mechanisms to understand the etiology of MS. Concomitantly, the use of a broader range of pre-clinical animal models for characterizing existing efficacious clinical treatments may elucidate additional or unexpected mechanisms of action for these drugs that augment insight into MS etiology. Herein, we explore the in vivo mechanism of action of dimethyl fumarate, which has been shown to suppress oxidative stress and immune cell responses in psoriasis and MS. Rather than studying this compound in the context of an experimental autoimmune-induced attack on the CNS, we have used a genetic model of hypomyelination, male rumpshaker (rsh) mice, which exhibit oligodendrocyte metabolic stress and startle-induced subcortical myoclonus during development and into adulthood. We find that myoclonus is reduced 30-50% in treated mutants but we do not detect substantial changes in metabolic or oxidative stress response pathways, cytokine modulation, or myelin thickness (assessed by anova). All procedures involving vertebrate animals in this study were reviewed and approved by the IACUC committee at Wayne State University.

Overexpression of CHOP in Myelinating Cells Does Not Confer a Significant Phenotype under Normal or Metabolic Stress Conditions.

UNLABELLED: The PKR-like endoplasmic reticulum kinase (PERK) pathway of the unfolded protein response (UPR) is protective against toxic accumulations of misfolded proteins in the endoplasmic reticulum, but is thought to drive cell death via the transcription factor, CHOP. However, in many cell types, CHOP is an obligate step in the PERK pathway, which frames the conundrum of a prosurvival pathway that kills cells. Our laboratory and others have previously demonstrated the prosurvival activity of the PERK pathway in oligodendrocytes. In the current study, we constitutively overexpress CHOP in myelinating cells during development and into adulthood under normal or UPR conditions. We show that this transcription factor does not drive apoptosis. Indeed, we observe no detriment in mice at multiple levels from single cells to mouse behavior and life span. In light of these data and other studies, we reinterpret PERK pathway function in the context of a stochastic vulnerability model, which governs the likelihood that cells undergo cell death upon cessation of UPR protection and while attempting to restore homeostasis. SIGNIFICANCE STATEMENT: Herein, we tackle the biggest controversy in the UPR literature: the function of the transcription factor CHOP as a protective or a prodeath factor. This manuscript is timely in light of the 2014 Lasker award for the UPR. Our in vivo data show that CHOP is not a prodeath protein, and we demonstrate that myelinating glial cells function normally in the presence of high CHOP expression from development to adulthood. Further, we propose a simplified view of UPR-mediated cell death after CHOP induction. We anticipate our work may turn the tide of the dogmatic view of CHOP and cause a reinvestigation of its function in different cell types. Accordingly, we believe our work will be a watershed for the UPR field.

Potential For Cell-mediated Immune Responses In Mouse Models Of Pelizaeus-Merzbacher Disease.

Although activation of the innate and adaptive arms of the immune system are undoubtedly involved in the pathophysiology of neurodegenerative diseases, it is unclear whether immune system activation is a primary or secondary event. Increasingly, published studies link primary metabolic stress to secondary inflammatory responses inside and outside of the nervous system. In this study, we show that the metabolic stress pathway known as the unfolded protein response (UPR) leads to secondary activation of the immune system. First, we observe innate immune system activation in autopsy specimens from Pelizaeus-Merzbacher disease (PMD) patients and mouse models stemming from PLP1 gene mutations. Second, missense mutations in mildly- and severely-affected Plp1-mutant mice exhibit immune-associated expression profiles with greater disease severity causing an increasingly proinflammatory environment. Third and unexpectedly, we find little evidence for dysregulated expression of major antioxidant pathways, suggesting that the unfolded protein and oxidative stress responses are separable. Together, these data show that UPR activation can precede innate and/or adaptive immune system activation and that neuroinflammation can be titrated by metabolic stress in oligodendrocytes. Whether-or-not such activation leads to autoimmune disease in humans is unclear, but the case report of steroid-mitigated symptoms in a PMD patient initially diagnosed with multiple sclerosis lends support.

Tissue-restricted transcription from a conserved intragenic CpG island in the Klf1 gene in mice.

Beyond Mendelian inheritance, an understanding of the complexities and consequences of the transfer of nonhereditary information to successive generations is at an early stage. Such epigenetic functionality is exemplified by DNA methylation and, as genome-wide high-throughput methodologies emerge, is increasingly being considered in the context of conserved intragenic and intergenic CpG islands that function as alternate sites of transcription initiation. Here we characterize an intragenic CpG island in exon 2 of the protein-coding mouse Klf1 gene, from which clustered transcription initiation sites yield positive-strand, severely truncated, capped and spliced RNAs. Expression from this CpG island in the testis begins between Postnatal Days 14-20, increases during development, and is temporally correlated with the maturation of secondary spermatocytes as they become the dominant cell population in the seminiferous epithelium. Only full-length KLF1-encoding mRNAs are detected in the hematopoietic tissue, spleen; thus, expression from the exon 2 CpG island is both developmentally regulated and tissue restricted. DNA methylation analysis indicates that spatiotemporal expression from the Klf1 CpG island is not associated with hypermethylation. Finally, our computational analysis from multiple species confirms intragenic transcription initiation and indicates that the KLF1 CpG island is evolutionarily conserved. Currently we have no evidence that these truncated RNAs can be translated via nonconventional mechanisms such as in-frame, conserved non-AUG-dependent Kozak consensus sequences; however, high-quality carboxyl-terminal antibodies will more effectively address this issue.

MpzR98C arrests Schwann cell development in a mouse model of early-onset Charcot-Marie-Tooth disease type 1B.

Mutations in myelin protein zero (MPZ) cause Charcot-Marie-Tooth disease type 1B. Many dominant MPZ mutations, including R98C, present as infantile onset dysmyelinating neuropathies. We have generated an R98C 'knock-in' mouse model of Charcot-Marie-Tooth type 1B, where a mutation encoding R98C was targeted to the mouse Mpz gene. Both heterozygous (R98C/+) and homozygous (R98C/R98C) mice develop weakness, abnormal nerve conduction velocities and morphologically abnormal myelin; R98C/R98C mice are more severely affected. MpzR98C is retained in the endoplasmic reticulum of Schwann cells and provokes a transitory, canonical unfolded protein response. Ablation of Chop, a mediator of the protein kinase RNA-like endoplasmic reticulum kinase unfolded protein response pathway restores compound muscle action potential amplitudes of R98C/+ mice but does not alter the reduced conduction velocities, reduced axonal diameters or clinical behaviour of these animals. R98C/R98C Schwann cells are developmentally arrested in the promyelinating stage, whereas development is delayed in R98C/+ mice. The proportion of cells expressing c-Jun, an inhibitor of myelination, is elevated in mutant nerves, whereas the proportion of cells expressing the promyelinating transcription factor Krox-20 is decreased, particularly in R98C/R98C mice. Our results provide a potential link between the accumulation of MpzR98C in the endoplasmic reticulum and a developmental delay in myelination. These mice provide a model by which we can begin to understand the early onset dysmyelination seen in patients with R98C and similar mutations.

Transgene-mediated rescue of spermatogenesis in Cldn11-null mice.

Claudins comprise a large family of tight junction (TJ) proteins that are often expressed broadly during development and in adult tissues and constitute the physical barriers that occlude the paracellular space in polarized epithelia. In mouse testis, the integrity of TJs is critical to normal spermatogenesis and is dependent on CLDN11 expression. In the current study, we have generated multiple transgenic mouse lines in which steady-state levels of transgene-derived Cldn11 mRNA are up to fourfold greater than endogenous gene expression. Spermatogenesis in all founder mice harboring two copies of the endogenous Cldn11 gene is normal. These animals breed well, indicating that transgene overexpression, at least at the level of mRNA, is well tolerated by Sertoli cells. In addition, we demonstrate that the promoter/enhancer of the transgene, comprising 5 kb of genomic sequence upstream of exon 1 of the mouse Cldn11 gene, is sufficient to rescue azoospermia in Cldn11-null mice. Finally, using transient transgenic mice, we narrow the location of Sertoli cell-specific cis regulatory elements to a 2-kb region upstream of the Cldn11 transcription start site. Together, these data provide essential information for further investigation of the biological regulation of CLDN11 TJs in the testis.

Microtubule deacetylases, SirT2 and HDAC6, in the nervous system.

Examination of the cytoskeleton has demonstrated the pivotal role of regulatory proteins governing cytoskeletal dynamics. Most work has focused on cell cycle and cell migration regarding cancer. However, these studies have yielded tremendous insight for development, particularly in the nervous system where all major cell types remodel their shape, generate unsurpassed quantities of membranes and extend cellular processes to communicate, and regulate the activities of other cells. Herein, we analyze two microtubule regulatory alpha-tubulin deacetylases, histone deacetylase-6 (HDAC6) and SirT2. HDAC6 is expressed by most neurons but is abundant in cerebellar Purkinje cells. In contrast, SirT2 is targeted to myelin sheaths. Expression of these proteins by post-mitotic cells indicates novel functions, such as process outgrowth and membrane remodeling. In oligodendrocytes, targeting of SirT2 to paranodes coincides with the presence of the microtubule-destabilizing protein stathmin-1 during early myelinogenesis and suggests the existence of a microtubule regulatory network that modulates cytoskeletal dynamics.

Novel alternatively spliced endoplasmic reticulum retention signal in the cytoplasmic loop of Proteolipid Protein-1.

Increased awareness about the importance of protein folding and trafficking to the etiology of gain-of-function diseases has driven extensive efforts to understand the cell and molecular biology underlying the life cycle of normal secretory pathway proteins and the detrimental effects of abnormal proteins. In this regard, the quality-control machinery in the endoplasmic reticulum (ER) has emerged as a major mechanism by which cells ensure that secreted and transmembrane proteins either adopt stable secondary, tertiary, and quaternary structures or are retained in the ER and degraded. Here we examine cellular and molecular aspects of ER retention in transfected fibroblasts expressing missense mutations in the Proteolipid Protein-1 (PLP1) gene that cause mild or severe forms of neurodegenerative disease in humans. Mild mutations cause protein retention in the ER that is partially dependent on the presence of a cytoplasmically exposed heptapeptide, KGRGSRG. In contrast, retention associated with severe mutations occurs independently of this peptide. Accordingly, the function of this novel heptapeptide has a significant impact on pathogenesis and provides new insight into the functions of the two splice isoforms encoded by the PLP1 gene, PLP1 and DM-20.

CNS myelin paranodes require Nkx6-2 homeoprotein transcriptional activity for normal structure.

Homeodomain proteins play critical roles during development in cell fate determination and proliferation, but few studies have defined gene regulatory networks for this class of transcription factors in differentiated cells. Using a lacZ-knock-in strategy to ablate Nkx6-2, we find that the Nkx6-2 promoter is active embryonically in neuroblasts and postnatally in oligodendrocytes. In addition to neurological deficits, we find widespread ultrastructural abnormalities in CNS white matter and aberrant expression of three genes encoding a paranodal microtubule destabilizing protein, stathmin 1, and the paranodal cell adhesion molecules neurofascin and contactin. The involvement of these downstream proteins in cytoskeletal function and cell adhesion reveals mechanisms whereby Nkx6-2 directly or indirectly regulates axon- glial interactions at myelin paranodes. Nkx6-2 does not appear to be the central regulator of axoglial junction assembly; nonetheless, our data constitute the first evidence of such a regulatory network and provide novel insights into the mechanism and effector molecules that are involved.

Deafness in Claudin 11-null mice reveals the critical contribution of basal cell tight junctions to stria vascularis function.

Generation of a strong electrical potential in the cochlea is uniquely mammalian and may reflect recent evolutionary advances in cellular voltage-dependent amplifiers. This endocochlear potential is hypothesized to dramatically improve hearing sensitivity, a concept that is difficult to explore experimentally, because manipulating cochlear function frequently causes rapid degenerative changes early in development. Here, we examine the deafness phenotype in adult Claudin 11-null mice, which lack the basal cell tight junctions that give rise to the intrastrial compartment and find little evidence of cochlear pathology. Potassium ion recycling is normal in these mutants, but endocochlear potentials were below 30 mV and hearing thresholds were elevated 50 dB sound pressure level across the frequency spectrum. Together, these data demonstrate the central importance of basal cell tight junctions in the stria vascularis and directly verify the two-cell hypothesis for generation of endocochlear potential. Furthermore, these data indicate that endocochlear potential is an essential component of the power source for the mammalian cochlear amplifier.

Alternative promoters and polyadenylation regulate tissue-specific expression of Hemogen isoforms during hematopoiesis and spermatogenesis.

Hemogen is a nuclear protein encoded by HEMGN (also known as hemogen in mouse, EDAG in human and RP59 in rat). It is considered to be a hematopoiesis-specific gene that is expressed during the ontogeny of hematopoiesis. Herein, we characterize two distinct splicing variants of HEMGN mRNA with restricted expression to hematopoietic cells and to round spermatids in the testis, respectively. Expression of the testis-specific HEMGN mRNA (HEMGN-t) is developmentally regulated and is concurrent with the first wave of meiosis in prepuberal mice. Sequence analysis reveals that HEMGN-t and the hematopoietic HEMGN mRNA (HEMGN-h) share a common coding sequence with distinct 5' and 3' untranslated regions and that these two isoforms are transcribed from the same gene locus, HEMGN, through the use of alternative promoters and polyadenylation sites. Thus, HEMGN expression exemplifies a developmental regulatory mechanism by which the diversification of gene expression is achieved through using distinct regulatory sequences in different cell types. Moreover, the existence of a testis-specific isoform of HEMGN suggests a role in spermatogenesis. Finally, fluorescence in situ hybridization demonstrates that HEMGN is localized to chromosome 4 A5-B2 in mouse and to chromosome 9q22 in human, which is a region known to harbor a cluster of leukemia breakpoints.

The unfolded protein response modulates disease severity in Pelizaeus-Merzbacher disease.

The unfolded protein response (UPR) is a eukaryotic signaling pathway linking protein flux through the endoplasmic reticulum to transcription and translational repression. Herein, we demonstrate UPR activation in the leukodystrophy Pelizaeus-Merzbacher disease (PMD) as well as in three mouse models of this disease and transfected fibroblasts expressing mutant protein. The CHOP protein, widely known as a proapoptotic transcription factor, modulates pathogenesis in the mouse models of PMD; however, this protein exhibits antiapoptotic activity. Together, these data show that the UPR has the potential to modulate disease severity in many cells expressing mutant secretory pathway proteins. Thus, PMD represents the first member of a novel class of disparate degenerative diseases for which UPR activation and signaling is the common pathogenic mechanism.