Nascent Aß42 Fibrillization in Synaptic Endosomes Precedes Plaque Formation in a Mouse Model of Alzheimer's-like ß-Amyloidosis.

Accumulation of amyloid-ß peptide (Aß) aggregates in synapses may contribute to the profound synaptic loss characteristic of Alzheimer's disease (AD). The origin of synaptic Aß aggregates remains elusive, but loss of endosomal proteostasis may trigger their formation. In this study, we identified the synaptic compartments where Aß accumulates, and performed a longitudinal analysis of synaptosomes isolated from brains of TgCRND8 APP transgenic mice of either sex. To evaluate the specific contribution of Aß-degrading protease endothelin-converting enzyme (ECE-1) to synaptic/endosomal Aß homeostasis, we analyzed the effect of partial Ece1 KO in brain and complete ECE1 KO in SH-SY5Y cells. Global inhibition of ECE family members was used to further assess their role in preventing synaptic Aß accumulation. Results showed that, before extracellular amyloid deposition, synapses were burdened with detergent-soluble Aß monomers, oligomers, and fibrils. Levels of all soluble Aß species declined thereafter, as Aß42 turned progressively insoluble and accumulated in Aß-producing synaptic endosomal vesicles with characteristics of multivesicular bodies. Accordingly, fibrillar Aß was detected in brain exosomes. ECE-1-deficient mice had significantly increased endogenous synaptosomal Aß42 levels, and protease inhibitor experiments showed that, in TgCRND8 mice, synaptic Aß42 became nearly resistant to degradation by ECE-related proteases. Our study supports that Aß accumulating in synapses is produced locally, within endosomes, and does not require the presence of amyloid plaques. ECE-1 is a determinant factor controlling the accumulation and fibrillization of nascent Aß in endosomes and, in TgCRND8 mice, Aß overproduction causes rapid loss of Aß42 solubility that curtails ECE-mediated degradation.SIGNIFICANCE STATEMENT Deposition of aggregated Aß in extracellular plaques is a defining feature of AD. Aß aggregates also accumulate in synapses and may contribute to the profound synaptic loss and cognitive dysfunction typical of the disease. However, it is not clear whether synaptotoxic Aß is mainly derived from plaques or if it is produced and aggregated locally, within affected synaptic compartments. Filling this knowledge gap is important for the development of an effective treatment for AD, as extracellular and intrasynaptic pools of Aß may not be equally modulated by immunotherapies or other therapeutic approaches. In this manuscript, we provide evidence that Aß aggregates building up in synapses are formed locally, within synaptic endosomes, because of disruptions in nascent Aß proteostasis.

PARK7 Catalyzes Stereospecific Detoxification of Methylglyoxal Consistent with Glyoxalase and Not Deglycase Function.

The protein PARK7 (also known as DJ-1) has been implicated in several diseases, with the most notable being Parkinson's disease. While several molecular and cellular roles have been ascribed to DJ-1, there is no real consensus on what its true cellular functions are and how the loss of DJ-1 function may contribute to the pathogenesis of Parkinson's disease. Recent reports have implicated DJ-1 in the detoxification of several reactive metabolites that are produced during glycolytic metabolism, with the most notable being the α-oxoaldehyde species methylglyoxal. While it is generally agreed that DJ-1 is able to metabolize methylglyoxal to lactate, the mechanism by which it does so is hotly debated with potential implications for cellular function. In this work, we provide definitive evidence that recombinant DJ-1 produced in human cells prevents the stable glycation of other proteins through the conversion of methylglyoxal or a related alkynyl dicarbonyl probe to their corresponding α-hydroxy carboxylic acid products. This protective action of DJ-1 does not require a physical interaction with a target protein, providing direct evidence for a glutathione-free glyoxalase and not a deglycase mechanism of methylglyoxal detoxification. Stereospecific liquid chromatography-mass spectrometry (LC-MS) measurements further uncovered the existence of nonenzymatic production of racemic lactate from MGO under physiological buffer conditions, whereas incubation with DJ-1 predominantly produces l-lactate. Collectively, these studies provide direct support for the stereospecific conversion of MGO to l-lactate by DJ-1 in solution with negligible or no contribution of direct protein deglycation.

Brain Targeted AAV1-GALC Gene Therapy Reduces Psychosine and Extends Lifespan in a Mouse Model of Krabbe Disease.

Krabbe disease (KD) is a progressive and devasting neurological disorder that leads to the toxic accumulation of psychosine in the white matter of the central nervous system (CNS). The condition is inherited via biallelic, loss-of-function mutations in the galactosylceramidase (GALC) gene. To rescue GALC gene function in the CNS of the twitcher mouse model of KD, an adeno-associated virus serotype 1 vector expressing murine GALC under control of a chicken ß-actin promoter (AAV1-GALC) was administered to newborn mice by unilateral intracerebroventricular injection. AAV1-GALC treatment significantly improved body weight gain and survival of the twitcher mice (n = 8) when compared with untreated controls (n = 5). The maximum weight gain after postnatal day 10 was significantly increased from 81% to 217%. The median lifespan was extended from 43 days to 78 days (range: 74-88 days) in the AAV1-GALC-treated group. Widespread expression of GALC protein and alleviation of KD neuropathology were detected in the CNS of the treated mice when examined at the moribund stage. Functionally, elevated levels of psychosine were completely normalized in the forebrain region of the treated mice. In the posterior region, which includes the mid- and the hindbrain, psychosine was reduced by an average of 77% (range: 53-93%) compared to the controls. Notably, psychosine levels in this region were inversely correlated with body weight and lifespan of AAV1-GALC-treated mice, suggesting that the degree of viral transduction of posterior brain regions following ventricular injection determined treatment efficacy on growth and survivability, respectively. Overall, our results suggest that viral vector delivery via the cerebroventricular system can partially correct psychosine accumulation in brain that leads to slower disease progression in KD.

Widespread, Reversible Cysteine Modification by Methylglyoxal Regulates Metabolic Enzyme Function.

Methylglyoxal (MGO), a reactive metabolite byproduct of glucose metabolism, is known to form a variety of posttranslational modifications (PTMs) on nucleophilic amino acids. For example, cysteine, the most nucleophilic proteinogenic amino acid, forms reversible hemithioacetal and stable mercaptomethylimidazole adducts with MGO. The high reactivity of cysteine toward MGO and the rate of formation of such modifications provide the opportunity for mechanisms by which proteins and pathways might rapidly sense and respond to alterations in levels of MGO. This indirect measure of alterations in glycolytic flux would thereby allow disparate cellular processes to dynamically respond to changes in nutrient availability and utilization. Here we report the use of quantitative LC-MS/MS-based chemoproteomic profiling approaches with a cysteine-reactive probe to map the proteome-wide landscape of MGO modification of cysteine residues. This approach led to the identification of many sites of potential functional regulation by MGO. We further characterized the role that such modifications have in a catalytic cysteine residue in a key metabolic enzyme and the resulting effects on cellular metabolism.

Galactosylceramidase deficiency and pathological abnormalities in cerebral white matter of Krabbe disease.

Krabbe Disease (KD) is an autosomal recessive disorder that results from loss-of-function mutations in the GALC gene, which encodes lysosomal enzyme galactosylceramidase (GALC). Functional deficiency of GALC is toxic to myelin-producing cells, which leads to progressive demyelination in both the central and peripheral nervous systems. It is hypothesized that accumulation of psychosine, which can only be degraded by GALC, is a primary initiator of pathologic cascades. Despite the central role of GALC in KD pathomechanism, investigations of GALC deficiency at a protein level are largely absent, due in part, to the lack of sensitive antibodies in the field. Leveraging two custom antibodies that can detect GALC at endogenous levels, we demonstrated that GALC protein is predominantly localized to oligodendrocytes in cerebral white matter of an infant brain, consistent with its functional role in myelination. Mature GALC could also be quantitatively detected as a 26 kDa band by western blotting and correlated to enzyme activity in brain tissues. The p.Ile562Thr polymorphic variant, which is over-represented in the KD population, was associated with reduced mature GALC protein and activity. In three infantile KD cases, homozygous null mutations in GALC lead to deficiency in total GALC protein and activity. Interestingly, although GALC activity was absent, normal levels of total GALC protein were detected by a sandwich ELISA using our custom antibodies in a later-onset KD brain, which suggests that the assay has the potential to differentiate infantile- and later-onset KD cases. Among the infantile KD cases, we quantified a 5-fold increase in psychosine levels, and observed increased levels of acid ceramidase, a key enzyme for psychosine production, and hyperglycosylated lysosomal-associated membrane protein 1, a marker for lysosomal activation, in periventricular white matter, a major pathological brain region, when compared with age-matched normal controls. While near complete demyelination was observed in these cases, we quantified that an early-infantile case (age of death at 10 months) had about 3-fold increases in both globoid cells, a pathological hallmark for KD, and CD8-positive T lymphocytes, a pathological marker for multiple sclerosis, in the white matter when compared with a slower progressing infantile case (age of death at 21 months), which suggests a positive correlation between clinical severity and neuropathology. Taken together, our findings have advanced the understanding of GALC protein biology in the context of normal and KD brain white matter. We also revealed new neuropathological changes that may provide insights to understand KD pathogenesis.

Suppression of Bile Acid Synthesis in a Preterm Infant Receiving Prolonged Parenteral Nutrition.

Bile acid metabolism is altered in neonates on parenteral nutrition (PN), predisposing them to parenteral nutrition-associated liver disease. Cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in the bile acid synthesis pathway, is repressed by fibroblast growth factor 19 (FGF19) and phytosterols (PS). We describe a case of a preterm infant who developed necrotizing enterocolitis (NEC) and received exclusive PN for over 2 months. Our objective was to serially assess CYP7A1 activity and plasma FGF19 and PS concentrations in this infant case compared to five healthy preterm infants. We found that CYP7A1 activity increased during the first 2 weeks of life in control infants but was undetectable in the infant case. FGF19 concentrations were high at birth in all infants and subsequently declined and did not differ between the case and control infants. As expected, PS concentrations were elevated in the infant case and continued to increase despite lipid minimization. In conclusion, CYP7A1 activity was gradually upregulated in healthy preterm infants but remained suppressed in the infant requiring prolonged PN. Preterm infants also had elevated FGF19 concentrations at birth, which decreased with advancing postnatal age.

Microglial lysosome dysfunction contributes to white matter pathology and TDP-43 proteinopathy in GRN-associated FTD.

Loss-of-function mutations in the progranulin gene (GRN), which encodes progranulin (PGRN), are a major cause of frontotemporal dementia (FTD). GRN-associated FTD is characterized by TDP-43 inclusions and neuroinflammation, but how PGRN loss causes disease remains elusive. We show that Grn knockout (KO) mice have increased microgliosis in white matter and an accumulation of myelin debris in microglial lysosomes in the same regions. Accumulation of myelin debris is also observed in white matter of patients with GRN-associated FTD. In addition, our findings also suggest that PGRN insufficiency in microglia leads to impaired lysosomal-mediated clearance of myelin debris. Finally, Grn KO mice that are deficient in cathepsin D (Ctsd), a key lysosomal enzyme, have augmented myelin debris and increased neuronal TDP-43 pathology. Together, our data strongly imply that PGRN loss affects microglial activation and lysosomal function, resulting in the accumulation of myelin debris and contributing to TDP-43 pathology.

Developmental regulation of the gut-liver (FGF19-CYP7A1) axis in neonates.

Introduction: Fibroblast growth factor 19 (FGF19) is a gut-derived hormone that regulates the expression of CYP7A1, the rate-limiting enzyme in bile acid (BA) synthesis pathway. Dysregulation of the FGF19-CYP7A1 (gut-liver) axis is associated with cholestatic liver disease. Infants, especially preterm infants and those with intestinal failure are at high risk for developing cholestatic liver disease. The activity of the gut-liver axis has not been characterized in this population. Our objective was to assess relationships between circulating FGF19 concentrations and CYP7A1 activity in neonates.Materials and methods: Plasma samples were obtained longitudinally from term and preterm infants (22-41-week gestation) hospitalized in a neonatal intensive care unit. Infants with congenital and acquired gastrointestinal disorders were excluded. Plasma levels of 7α-hydroxy-4-cholesten-3-one (C4), a marker of CYP7A1 activity, were quantified using HPLC-MS/MS. Plasma FGF19 concentrations were quantified by ELISA. Data were analyzed using linear regression models and structural equation modeling.Results: One hundred eighty-one plasma samples were analyzed from 62 infants. C4 concentrations were undetectable prior to 30 weeks' gestation and, thereafter, increased with advancing gestational age and with volume of enteral feeds. They did not correlate with serum FGF19 concentrations, which decreased with advancing gestational age and volume of enteral feeds.Discussion: The activity of CYP7A1, the rate-limiting BA synthetic enzyme in adults, is developmentally regulated and undetectable in newborns less than 30 weeks' gestation. FGF19 concentrations do not correlate with CYP7A1 activity, suggesting that the gut-liver axis is not functional in infants. High FGF19 concentrations at birth in infants less than 37 weeks' gestation is a novel finding, and its source and role in preterm infants warrants further investigation.Rationale: The intestinal hormone, fibroblast growth factor 19 (FGF19), is a major regulator of CYP7A1, the rate limiting enzyme in bile acid (BA) synthesis. Recently, dysregulation of the gut-liver (FGF19-CYP7A1) axis has been implicated in adult cholestatic liver disease, and animal studies have shown that exogenous FGF19 protects against liver injury. Given the therapeutic potential related to this signaling pathway, we sought to characterize the association between CYP7A1 and FGF19 in term and preterm infants. We conducted a prospective, observational study that measured in vivo CYP7A1 activity and FGF19 concentrations in 62 term and preterm infants (n = 181 samples). We found that CYP7A1 activity is developmentally regulated; its activity is undetectable prior to 30 weeks' gestation and increases with advancing gestational age and volume of enteral feeds. Contrary to expectation, we demonstrated that FGF19 is expressed at birth in preterm infants and decreases over time, even as enteral feeds increase. Using structural equation modeling, we were able to show that CYP7A1 activity does not correlate with FGF19 concentrations. Our results suggest that the gut-liver axis is not upregulated in preterm and term infants and that neonates with cholestatic liver disease will unlikely benefit from supplemental FGF19. We also report novel findings of elevated FGF19 concentrations in preterm infants at birth and speculate that there may be an extra-intestinal source of FGF19 that is developmentally expressed in these infants.

Mouse Models of C9orf72 Hexanucleotide Repeat Expansion in Amyotrophic Lateral Sclerosis/ Frontotemporal Dementia.

The presence of hexanucleotide repeat expansion (HRE) in the first intron of the human C9orf72 gene is the most common genetic cause underlying both familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Studies aimed at elucidating the pathogenic mechanisms associated of C9orf72 FTD and ALS (C9FTD/ALS) have focused on the hypothesis of RNA and protein toxic gain-of-function models, including formation of nuclear RNA foci containing GGGGCC (G4C2) HRE, inclusions containing dipeptide repeat proteins through a non-canonical repeat associated non-ATG (RAN) translation mechanism, and on loss-of-function of the C9orf72 protein. Immense effort to elucidate these mechanisms has been put forth and toxic gain-of-function models have especially gained attention. Various mouse models that recapitulate distinct disease-related pathological, functional, and behavioral phenotypes have been generated and characterized. Although these models express the C9orf72 HRE mutation, there are numerous differences among them, including the transgenesis approach to introduce G4C2-repeat DNA, genomic coverage of C9orf72 features in the transgene, G4C2-repeat length after genomic stabilization, spatiotemporal expression profiles of RNA foci and RAN protein aggregates, neuropathological features, and neurodegeneration-related clinical symptoms. This review aims to (1) provide an overview of the key characteristics; (2) provide insights into potential pathological factors contributing to neurotoxicity and clinical phenotypes through systematic comparison of these models.

The lysosomal protein cathepsin L is a progranulin protease.

Haploinsufficiency of GRN, the gene encoding progranulin (PGRN), causes frontotemporal lobar degeneration (FTLD), the second most common cause of early-onset dementia. Receptor-mediated lysosomal targeting has been shown to regulate brain PGRN levels, and complete deficiency of PGRN is a direct cause of neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. Here we show that the lysosomal cysteine protease cathepsin L (Cat L) can mediate the proteolytic cleavage of intracellular PGRN into poly-granulin and granulin fragments. Further, PGRN and Cat L co-localize in lysosomes of HEK293 cells, iPSC-derived neurons and human cortical neurons from human postmortem tissue. These data identify Cat L as a key intracellular lysosomal PGRN protease, and provides an intriguing new link between lysosomal dysfunction and FTLD.

Poly(GP) proteins are a useful pharmacodynamic marker for C9ORF72-associated amyotrophic lateral sclerosis.

There is no effective treatment for amyotrophic lateral sclerosis (ALS), a devastating motor neuron disease. However, discovery of a G4C2 repeat expansion in the C9ORF72 gene as the most common genetic cause of ALS has opened up new avenues for therapeutic intervention for this form of ALS. G4C2 repeat expansion RNAs and proteins of repeating dipeptides synthesized from these transcripts are believed to play a key role in C9ORF72-associated ALS (c9ALS). Therapeutics that target G4C2 RNA, such as antisense oligonucleotides (ASOs) and small molecules, are thus being actively investigated. A limitation in moving such treatments from bench to bedside is a lack of pharmacodynamic markers for use in clinical trials. We explored whether poly(GP) proteins translated from G4C2 RNA could serve such a purpose. Poly(GP) proteins were detected in cerebrospinal fluid (CSF) and in peripheral blood mononuclear cells from c9ALS patients and, notably, from asymptomatic C9ORF72 mutation carriers. Moreover, CSF poly(GP) proteins remained relatively constant over time, boding well for their use in gauging biochemical responses to potential treatments. Treating c9ALS patient cells or a mouse model of c9ALS with ASOs that target G4C2 RNA resulted in decreased intracellular and extracellular poly(GP) proteins. This decrease paralleled reductions in G4C2 RNA and downstream G4C2 RNA-mediated events. These findings indicate that tracking poly(GP) proteins in CSF could provide a means to assess target engagement of G4C2 RNA-based therapies in symptomatic C9ORF72 repeat expansion carriers and presymptomatic individuals who are expected to benefit from early therapeutic intervention.

Progranulin gene variation affects serum progranulin levels differently in Danish bipolar individuals compared with healthy controls.

OBJECTIVES: The identification of peripheral biomarkers for bipolar disorder is of great importance and has the potential to improve diagnosis, treatment and prognosis. Recent studies have reported lower plasma progranulin levels in bipolar individuals compared with controls and association with single nucleotide polymorphisms (SNPs) within the progranulin gene (GRN). In the present study, we investigated the effect of GRN and sortilin (SORT1) gene variation on serum progranulin levels in bipolar individuals and controls. MATERIALS AND METHODS: In a Danish cohort of individuals with bipolar disorder and controls, we analysed the serum progranulin level (nbipolar=80, ncontrols=76) and five SNPs located within GRN and two SNPs near the SORT1 gene encoding sortilin, a progranulin scavenger receptor known to affect circulating progranulin levels (nbipolar=166, ncontrols=186). RESULTS: We observed no significant difference in the serum progranulin level between cases and controls and none of the analysed SNPs located within GRN or close to SORT1 were associated with bipolar disorder. Crude and adjusted (adjusted for case-control status, sex and age) linear regression analyses showed no effect of any SNPs on the serum progranulin level. However, we observed that the mean serum progranulin level in cases and controls is affected differently depending on the genotypes of two SNPs within GRN (rs2879096 and rs4792938). LIMITATION: The sample size is relatively small and detailed information on medication and polarity of the disorder is not available. No correction for multiple testing was performed. CONCLUSION: Our study suggests that the potential of progranulin as a biomarker for bipolar disorder is genotype dependent.

The extreme N-terminus of TDP-43 mediates the cytoplasmic aggregation of TDP-43 and associated toxicity in vivo.

Inclusions of Tar DNA- binding protein 43 (TDP-43) are a pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP). Pathological TDP-43 exhibits the disease-specific biochemical signatures, which include its ubiquitination, phosphorylation and truncation. Recently, we demonstrated that the extreme N-terminus of TDP-43 regulates formation of abnormal cytoplasmic TDP-43 aggregation in cultured cells and primary neurons. However, it remained unclear whether this N-terminal domain mediates TDP-43 aggregation and the associated toxicity in vivo. To investigate this, we expressed a GFP-tagged TDP-43 with a nuclear localization signal mutation (GFP-TDP-43NLSm) and a truncated form without the extreme N-terminus (GFP-TDP-4310-414-NLSm) by adeno-associated viral (AAV) vectors in mouse primary cortical neurons and murine central nervous system. Compared to neurons containing GFP alone, expression of GFP-TDP-43NLSm resulted in the formation of ubiquitin-positive cytoplasmic inclusions and activation of caspase-3, an indicator of cell death. Moreover, mice expressing GFP-TDP-43NLSm proteins show reactive gliosis and develop neurological abnormalities. However, by deletion of TDP-43's extreme N-terminus, these pathological alterations can be abrogated. Together, our study provides further evidence confirming the critical role of the extreme N-terminus of TDP-43 in regulating protein structure as well as mediating toxicity associated with its aggregation. This article is part of a Special Issue entitled SI:RNA Metabolism in Disease.

Neurodegeneration. C9ORF72 repeat expansions in mice cause TDP-43 pathology, neuronal loss, and behavioral deficits.

The major genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis is a G4C2 repeat expansion in C9ORF72. Efforts to combat neurodegeneration associated with "c9FTD/ALS" are hindered by a lack of animal models recapitulating disease features. We developed a mouse model to mimic both neuropathological and clinical c9FTD/ALS phenotypes. We expressed (G4C2)66 throughout the murine central nervous system by means of somatic brain transgenesis mediated by adeno-associated virus. Brains of 6-month-old mice contained nuclear RNA foci, inclusions of poly(Gly-Pro), poly(Gly-Ala), and poly(Gly-Arg) dipeptide repeat proteins, as well as TDP-43 pathology. These mouse brains also exhibited cortical neuron and cerebellar Purkinje cell loss, astrogliosis, and decreased weight. (G4C2)66 mice also developed behavioral abnormalities similar to clinical symptoms of c9FTD/ALS patients, including hyperactivity, anxiety, antisocial behavior, and motor deficits.