ZNF142 mutation causes sex-dependent neurologic disorder.

BACKGROUND: Sex-specific predilection in neurological diseases caused by mutations in autosomal genes is a phenomenon whose molecular basis is poorly understood. We studied females of consanguineous Bedouin kindred presenting with severe global developmental delay and epilepsy. METHODS: Linkage analysis, whole exome sequencing, generation of CRISPR/cas9 knock-in mice, mouse behaviour and molecular studies RESULTS: Linkage analysis and whole exome sequencing studies of the affected kindred delineated a ~5 Mbp disease-associated chromosome 2q35 locus, containing a novel homozygous frameshift truncating mutation in ZNF142, in line with recent studies depicting similar ZNF142 putative loss-of-function human phenotypes with female preponderance. We generated knock-in mice with a truncating mutation adjacent to the human mutation in the mouse ortholog. Behaviour studies of homozygous Zfp142R1508* mice showed significant phenotype only in mutant females, with learning and memory deficits, hyperactivity and aberrant loss of fear of open spaces. Bone marrow and spleen of homozygous Zfp142R1508* mice showed depletion of lymphoid and haematopoietic cells, mostly in females. RT-PCR showed lower expression of Zpf142 in brain compartments of female versus male wild-type mice. RNA-seq studies of hippocampus, hypothalamus, cortex and cerebellum of female wild-type versus homozygous Zfp142R1508* mice demonstrated differentially expressed genes. Notably, expression of Taok1 in the cortex and of Mllt6 in the hippocampus was downregulated in homozygous Zfp142R1508* mice. Taok1 mutations have been associated with aberrant neurodevelopment and behaviour. Mllt6 expression is regulated by sex hormones and Mllt6 null-mutant mice present with haematopoietic, immune system and female-specific behaviour phenotypes. CONCLUSION: ZNF142 mutation downregulates Mllt6 and Taok1, causing a neurodevelopmental phenotype in humans and mice with female preponderance.

CD4 T-cell aging exacerbates neuroinflammation in a late-onset mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive neurodegenerative disorder characterized by the loss of upper and lower motor neurons in the brain and spinal cord. Accumulating evidence suggests that ALS is not solely a neuronal cell- or brain tissue-autonomous disease and that neuroinflammation plays a key role in disease progression. Furthermore, whereas both CD4 and CD8 T cells were observed in spinal cords of ALS patients and in mouse models of the disease, their role in the neuroinflammatory process, especially considering their functional changes with age, is not fully explored. In this study, we revealed the structure of the CD4 T-cell compartment during disease progression of early-onset SOD1G93A and late-onset SOD1G37R mouse models of ALS. We show age-related changes in the CD4 T-cell subset organization between these mutant SOD1 mouse models towards increased frequency of effector T cells in spleens of SOD1G37R mice and robust infiltration of CD4 T cells expressing activation markers and the checkpoint molecule PD1 into the spinal cord. The frequency of infiltrating CD4 T cells correlated with the frequency of infiltrating CD8 T cells which displayed a more exhausted phenotype. Moreover, RNA-Seq and immunohistochemistry analyses of spinal cords from SOD1G37R mice with early clinical symptoms demonstrated immunological trajectories reminiscent of a neurotoxic inflammatory response which involved proinflammatory T cells and antigen presentation related pathways. Overall, our findings suggest that age-related changes of the CD4 T cell landscape is indicative of a chronic inflammatory response, which aggravates the disease process and can be therapeutically targeted.

Hyperinsulinism/hyperammonemia syndrome caused by biallelic SLC25A36 mutation.

Hyperinsulinism/hyperammonemia (HI/HA) syndrome has been known to be caused by dominant gain-of-function mutations in GLUD1, encoding the mitochondrial enzyme glutamate dehydrogenase. Pathogenic GLUD1 mutations enhance enzymatic activity by reducing its sensitivity to allosteric inhibition by GTP. Two recent independent studies showed that a similar HI/HA phenotype can be caused by biallelic mutations in SLC25A36, encoding pyrimidine nucleotide carrier 2 (PNC2), a mitochondrial nucleotide carrier that transports pyrimidine and guanine nucleotides across the inner mitochondrial membrane: one study reported a single case caused by a homozygous truncating mutation in SLC25A36 resulting in lack of expression of SLC25A36 in patients' fibroblasts. A second study described two siblings with a splice site mutation in SLC25A36, causing reduction of mitochondrial GTP content, putatively leading to hyperactivation of glutamate dehydrogenase. In an independent study, through combined linkage analysis and exome sequencing, we demonstrate in four individuals of two Bedouin Israeli related families the same disease-causing SLC25A36 (NM_018155.3) c.284 + 3A > T homozygous splice-site mutation found in the two siblings. We demonstrate that the mutation, while causing skipping of exon 3, does not abrogate expression of mRNA and protein of the mutant SLC25A36 in patients' blood and fibroblasts. Affected individuals had hyperinsulinism, hyperammonemia, borderline low birth weight, tonic-clonic seizures commencing around 6 months of age, yet normal intellect and no significant other morbidities. Chronic constipation, hypothyroidism, and developmental delay previously described in a single patient were not found. We thus verify that biallelic SLC25A36 mutations indeed cause HI/HA syndrome and clearly delineate the disease phenotype.

SEC31A mutation affects ER homeostasis, causing a neurological syndrome.

BACKGROUND: Consanguineous kindred presented with an autosomal recessive syndrome of intrauterine growth retardation, marked developmental delay, spastic quadriplegia with profound contractures, pseudobulbar palsy with recurrent aspirations, epilepsy, dysmorphism, neurosensory deafness and optic nerve atrophy with no eye fixation. Affected individuals died by the age of 4. Brain MRI demonstrated microcephaly, semilobar holoprosencephaly and agenesis of corpus callosum. We aimed at elucidating the molecular basis of this disease. METHODS: Genome-wide linkage analysis combined with whole exome sequencing were performed to identify disease-causing variants. Functional consequences were investigated in fruit flies null mutant for the Drosophila SEC31A orthologue. SEC31A knockout SH-SY5Y and HEK293T cell-lines were generated using CRISPR/Cas9 and studied through qRT-PCR, immunoblotting and viability assays. RESULTS: Through genetic studies, we identified a disease-associated homozygous nonsense mutation in SEC31A. We demonstrate that SEC31A is ubiquitously expressed, and that the mutation triggers nonsense-mediated decay of its transcript, comprising a practical null mutation. Similar to the human disease phenotype, knockdown SEC31A flies had defective brains and early lethality. Moreover, in line with SEC31A encoding one of the two coating layers comprising the Coat protein complex II (COP-II) complex, trafficking newly synthesised proteins from the endoplasmic reticulum (ER) to the Golgi, CRISPR/Cas9-mediated SEC31A null mutant cells demonstrated reduced viability through upregulation of ER-stress pathways. CONCLUSION: We demonstrate through human and Drosophila genetic and in vitro molecular studies, that a severe neurological syndrome is caused by a null mutation in SEC31A, reducing cell viability through enhanced ER-stress response, in line with SEC31A's role in the COP-II complex.

SIRT6 is a key regulator of mitochondrial function in the brain.

The SIRT6 deacetylase has been implicated in DNA repair, telomere maintenance, glucose and lipid metabolism and, importantly, it has critical roles in the brain ranging from its development to neurodegeneration. Here, we combined transcriptomics and metabolomics approaches to characterize the functions of SIRT6 in mouse brains. Our analysis reveals that SIRT6 is a central regulator of mitochondrial activity in the brain. SIRT6 deficiency in the brain leads to mitochondrial deficiency with a global downregulation of mitochondria-related genes and pronounced changes in metabolite content. We suggest that SIRT6 affects mitochondrial functions through its interaction with the transcription factor YY1 that, together, regulate mitochondrial gene expression. Moreover, SIRT6 target genes include SIRT3 and SIRT4, which are significantly downregulated in SIRT6-deficient brains. Our results demonstrate that the lack of SIRT6 leads to decreased mitochondrial gene expression and metabolomic changes of TCA cycle byproducts, including increased ROS production, reduced mitochondrial number, and impaired membrane potential that can be partially rescued by restoring SIRT3 and SIRT4 levels. Importantly, the changes we observed in SIRT6-deficient brains are also occurring in aging human brains and particularly in patients with Alzheimer's, Parkinson's, Huntington's, and Amyotrophic lateral sclerosis disease. Overall, our results suggest that the reduced levels of SIRT6 in the aging brain and neurodegeneration initiate mitochondrial dysfunction by altering gene expression, ROS production, and mitochondrial decay.

An optimized intracerebroventricular injection of CD4+ T cells into mice.

The blood-brain barrier acts as a major barrier for the entrance of most therapeutics into the brain, impeding treatment for neurological disorders. Intracerebroventricular (ICV) injection of T cells is a useful tool for cell therapy of neurological disorders including neurodegenerative and neuropsychiatric diseases and brain tumors. Here, we present an optimized ICV injection of T cells with improved injection efficiency at pathological sites within the brain parenchyma. We describe details of the surgical procedure and verification of injection via immunohistochemistry. For complete details on the use and execution of this protocol, please refer to Fisher et al. (2014); Strominger et al., (2018); Mittal et al. (2019); Eremenko et al. (2019).

FACS-based isolation of fixed mouse neuronal nuclei for ATAC-seq and Hi-C.

The organization of chromatin structure plays a crucial role in gene expression, DNA replication, and repair. Chromatin alterations influence gene expression, and modifications could be associated with genomic instability in the cells during aging or diseases. Here, we provide a modified protocol to isolate fixed neuronal nuclei from a single mouse cortex to investigate the spatial organization of chromatin structure on a genome-wide scale by ATAC-seq (the assay for transposase-accessible chromatin with high-throughput sequencing) and chromatin conformation by Hi-C (high-throughput chromosome conformation capture).

An optimized protocol for the retroviral transduction of mouse CD4 T cells.

Transduction of primary T cells has become prominent with the introduction of chimeric antigen receptor T-cell therapy. Although there are many protocols for the transduction of human T cells, it remains a challenge to transduce murine T cells. We present an optimized protocol for the retroviral transduction of murine CD4 T cells, which overcomes major challenges including large-scale production and long-term culturing of transduced cells. The optimized protocol combines high transduction efficiency with a low rate of cell death. For complete details on the use and execution of this protocol, please refer to Eremenko et al., 2019.

A Mechanism for the Inhibition of Tau Neurotoxicity: Studies with Artificial Membranes, Isolated Mitochondria, and Intact Cells.

It is currently believed that molecular agents that specifically bind to and neutralize the toxic proteins/peptides, amyloid ß (Aß42), tau, and the tau-derived peptide PHF6, hold the key to attenuating the progression of Alzheimer's disease (AD). We thus tested our previously developed nonaggregating Aß42 double mutant (Aß42DM) as a multispecific binder for three AD-associated molecules, wild-type Aß42, the tauK174Q mutant, and a synthetic PHF6 peptide. Aß42DM acted as a functional inhibitor of these molecules in in vitro assays and in neuronal cell-based models of AD. The double mutant bound both cytotoxic tauK174Q and synthetic PHF6 and protected neuronal cells from the accumulation of tau in cell lysates and mitochondria. Aß42DM also reduced toxic intracellular levels of calcium and the overall cell toxicity induced by overexpressed tau, synthetic PHF6, Aß42, or a combination of PHF6and Aß42. Aß42DM inhibited PHF6-induced overall mitochondrial dysfunction: In particular, Aß42DM inhibited PHF6-induced damage to submitochondrial particles (SMPs) and suppressed PHF6-induced elevation of the ζ-potential of inverted SMPs (proxy for the inner mitochondrial membrane, IMM). PHF6 reduced the lipid fluidity of cardiolipin/DOPC vesicles (that mimic the IMM) but not DOPC (which mimics the outer mitochondrial membrane), and this effect was inhibited by Aß42DM. This inhibition may be explained by the conformational changes in PHF6 induced by Aß42DM in solution and in membrane mimetics. On this basis, the paper presents a mechanistic explanation for the inhibitory activity of Aß42DM against Aß42- and tau-induced membrane permeability and cell toxicity and provides confirmatory evidence for its protective function in neuronal cells.

SIRT6-CBP-dependent nuclear Tau accumulation and its role in protein synthesis.

Several neurodegenerative diseases present Tau accumulation as the main pathological marker. Tau post-translational modifications such as phosphorylation and acetylation are increased in neurodegeneration. Here, we show that Tau hyper-acetylation at residue 174 increases its own nuclear presence and is the result of DNA damage signaling or the lack of SIRT6, both causative of neurodegeneration. Tau-K174ac is deacetylated in the nucleus by SIRT6. However, lack of SIRT6 or chronic DNA damage results in nuclear Tau-K174ac accumulation. Once there, it induces global changes in gene expression, affecting protein translation, synthesis, and energy production. Concomitantly, Alzheimer's disease (AD) case subjects show increased nucleolin and a decrease in SIRT6 levels. AD case subjects present increased levels of nuclear Tau, particularly Tau-K174ac. Our results suggest that increased Tau-K174ac in AD case subjects is the result of DNA damage signaling and SIRT6 depletion. We propose that Tau-K174ac toxicity is due to its increased stability, nuclear accumulation, and nucleolar dysfunction.

BDNF-producing, amyloid ß-specific CD4 T cells as targeted drug-delivery vehicles in Alzheimer's disease.

BACKGROUND: The delivery of therapeutic proteins to selected sites within the central nervous system (CNS) parenchyma is a major challenge in the treatment of various neurodegenerative disorders. As brain-derived neurotrophic factor (BDNF) is reduced in the brain of people with Alzheimer's disease (AD) and its administration has shown promising therapeutic effects in mouse model of the disease, we generated a novel platform for T cell-based BDNF delivery into the brain parenchyma. METHODS: We generated amyloid beta-protein (Aß)-specific CD4 T cells (Aß-T cells), genetically engineered to express BDNF, and injected them intracerebroventricularly into the 5XFAD mouse model of AD. FINDINGS: The BDNF-secreting Aß-T cells migrated efficiently to amyloid plaques, where they significantly increased the levels of BDNF, its receptor TrkB, and various synaptic proteins known to be reduced in AD. Furthermore, the injected mice demonstrated reduced levels of beta-secretase 1 (BACE1)-a protease essential in the cleavage process of the amyloid precursor protein-and ameliorated amyloid pathology and inflammation within the brain parenchyma. INTERPRETATION: A T cell-based delivery of proteins into the brain can serve as a platform to modulate neurotoxic inflammation and to promote neuronal repair in neurodegenerative diseases.

CD4 T Cells Induce A Subset of MHCII-Expressing Microglia that Attenuates Alzheimer Pathology.

Microglia play a key role in innate immunity in Alzheimer disease (AD), but their role as antigen-presenting cells is as yet unclear. Here we found that amyloid ß peptide (Aß)-specific T helper 1 (Aß-Th1 cells) T cells polarized to secrete interferon-γ and intracerebroventricularly (ICV) injected to the 5XFAD mouse model of AD induced the differentiation of major histocompatibility complex class II (MHCII)+ microglia with distinct morphology and enhanced plaque clearance capacity than MHCII- microglia. Notably, 5XFAD mice lacking MHCII exhibited an enhanced amyloid pathology in the brain along with exacerbated innate inflammation and reduced phagocytic capacity. Using a bone marrow chimera mouse model, we showed that infiltrating macrophages did not differentiate to MHCII+ cells following ICV injection of Aß-Th1 cells and did not support T cell-mediated amyloid clearance. Overall, we demonstrate that CD4 T cells induce a P2ry12+ MHCII+ subset of microglia, which play a key role in T cell-mediated effector functions that abrogate AD-like pathology.

Aging promotes reorganization of the CD4 T cell landscape toward extreme regulatory and effector phenotypes.

Age-associated changes in CD4 T-cell functionality have been linked to chronic inflammation and decreased immunity. However, a detailed characterization of CD4 T cell phenotypes that could explain these dysregulated functional properties is lacking. We used single-cell RNA sequencing and multidimensional protein analyses to profile thousands of CD4 T cells obtained from young and old mice. We found that the landscape of CD4 T cell subsets differs markedly between young and old mice, such that three cell subsets-exhausted, cytotoxic, and activated regulatory T cells (aTregs)-appear rarely in young mice but gradually accumulate with age. Most unexpected were the extreme pro- and anti-inflammatory phenotypes of cytotoxic CD4 T cells and aTregs, respectively. These findings provide a comprehensive view of the dynamic reorganization of the CD4 T cell milieu with age and illuminate dominant subsets associated with chronic inflammation and immunity decline, suggesting new therapeutic avenues for age-related diseases.

Aggregation of human S100A8 and S100A9 amyloidogenic proteins perturbs proteostasis in a yeast model.

Amyloid aggregates of the calcium-binding EF-hand proteins, S100A8 and S100A9, have been found in the corpora amylacea of patients with prostate cancer and may play a role in carcinogenesis. Here we present a novel model system using the yeast Saccharomyces cerevisiae to study human S100A8 and S100A9 aggregation and toxicity. We found that S100A8, S100A9 and S100A8/9 cotransfomants form SDS-resistant non-toxic aggregates in yeast cells. Using fluorescently tagged proteins, we showed that S100A8 and S100A9 accumulate in foci. After prolonged induction, S100A8 foci localized to the cell vacuole, whereas the S100A9 foci remained in the cytoplasm when present alone, but entered the vacuole in cotransformants. Biochemical analysis of the proteins indicated that S100A8 and S100A9 alone or coexpressed together form amyloid-like aggregates in yeast. Expression of S100A8 and S100A9 in wild type yeast did not affect cell viability, but these proteins were toxic when expressed on a background of unrelated metastable temperature-sensitive mutant proteins, Cdc53-1p, Cdc34-2p, Srp1-31p and Sec27-1p. This finding suggests that the expression and aggregation of S100A8 and S100A9 may limit the capacity of the cellular proteostasis machinery. To test this hypothesis, we screened a set of chaperone deletion mutants and found that reducing the levels of the heat-shock proteins Hsp104p and Hsp70p was sufficient to induce S100A8 and S100A9 toxicity. This result indicates that the chaperone activity of the Hsp104/Hsp70 bi-chaperone system in wild type cells is sufficient to reduce S100A8 and S100A9 amyloid toxicity and preserve cellular proteostasis. Expression of human S100A8 and S100A9 in yeast thus provides a novel model system for the study of the interaction of amyloid deposits with the proteostasis machinery.