Autophagic Abnormalities Research Articles

Autophagy: A Key Regulator of Homeostasis and Disease: An Overview of Molecular Mechanisms and Modulators.

Autophagy is a highly conserved lysosomal degradation pathway active at basal levels in all cells. However, under stress conditions, such as a lack of nutrients or trophic factors, it works as a survival mechanism that allows the generation of metabolic precursors for the proper functioning of the cells until the nutrients are available. Neurons, as post-mitotic cells, depend largely on autophagy to maintain cell homeostasis to get rid of damaged and/or old organelles and misfolded or aggregated proteins. Therefore, the dysfunction of this process contributes to the pathologies of many human diseases. Furthermore, autophagy is highly active during differentiation and development. In this review, we describe the current knowledge of the different pathways, molecular mechanisms, factors that induce it, and the regulation of mammalian autophagy. We also discuss its relevant role in development and disease. Finally, here we summarize several investigations demonstrating that autophagic abnormalities have been considered the underlying reasons for many human diseases, including liver disease, cardiovascular, cerebrovascular diseases, neurodegenerative diseases, neoplastic diseases, cancers, and, more recently, infectious diseases, such as SARS-CoV-2 caused COVID-19 disease.

A Novel Homozygous VPS11 Variant May Cause Generalized Dystonia.

In this work, we describe the association of a novel homozygous VPS11 variant with adult‐onset generalized dystonia, providing a detailed clinical report and biological evidence of disease mechanism. Vps11 is a subunit of the homotypic fusion and protein sorting (HOPS) complex, which promotes the fusion of late endosomes and autophagosomes with the lysosome. Functional studies on mutated fibroblasts showed marked lysosomal and autophagic abnormalities, which improved after overexpression of the wild type Vps11 protein. In conclusion, a deleterious VPS11 variant, damaging the autophagic and lysosomal pathways, is the probable genetic cause of a novel form of generalized dystonia. ANN NEUROL 2021;89:834–839

Mitochondrial translation deficiency impairs NAD+‐mediated lysosomal acidification

Mitochondrial translation dysfunction is associated with neurodegenerative and cardiovascular diseases. Cells eliminate defective mitochondria by the lysosomal machinery via autophagy. The relationship between mitochondrial translation and lysosomal function is unknown. In this study, mitochondrial translation‐deficient hearts from p32‐knockout mice were found to exhibit enlarged lysosomes containing lipofuscin, suggesting impaired lysosome and autolysosome function. These mice also displayed autophagic abnormalities, such as p62 accumulation and LC3 localization around broken mitochondria. The expression of genes encoding for nicotinamide adenine dinucleotide (NAD+) biosynthetic enzymes—Nmnat3 and Nampt—and NAD+ levels were decreased, suggesting that NAD+ is essential for maintaining lysosomal acidification. Conversely, nicotinamide mononucleotide (NMN) administration or Nmnat3 overexpression rescued lysosomal acidification. Nmnat3 gene expression is suppressed by HIF1α, a transcription factor that is stabilized by mitochondrial translation dysfunction, suggesting that HIF1α‐Nmnat3‐mediated NAD+ production is important for lysosomal function. The glycolytic enzymes GAPDH and PGK1 were found associated with lysosomal vesicles, and NAD+ was required for ATP production around lysosomal vesicles. Thus, we conclude that NAD+ content affected by mitochondrial dysfunction is essential for lysosomal maintenance.

Optineurin defects cause TDP43-pathology with autophagic vacuolar formation

We previously showed that optineurin (OPTN) mutations lead to the development of amyotrophic lateral sclerosis. The association between OPTN mutations and the pathogenesis of amyotrophic lateral sclerosis remains unclear. To investigate the mechanism underlying its pathogenesis, we generated Optn knockout mice. We evaluated histopathological observations of these mice and compared with those of OPTN- amyotrophic lateral sclerosis cases to investigate the mechanism underlying the pathogenesis of amyotrophic lateral sclerosis caused by OPTN mutations. The Optn (−/−) mice presented neuronal autophagic vacuoles immunopositive for charged multivesicular body protein 2b, one of the hallmarks of granulovacuolar degenerations, in the cytoplasm of spinal cord motor neurons at the age of 8 months and the OPTN- amyotrophic lateral sclerosis case with homozygous Q398X mutation. In addition, Optn (−/−) mice showed TAR-DNA binding protein 43/sequestosome1/p62 -positive cytoplasmic inclusions and the clearance of nuclear TAR-DNA binding protein 43. The axonal degeneration of the sciatic nerves was observed in Optn (−/−) mice. However, we could not observe significant differences in survival time, body weight, and motor functions, at 24 months. Our findings suggest that homozygous OPTN deletion or mutations might result in autophagic dysfunction and TAR-DNA binding protein 43 mislocalization, thereby leading to neurodegeneration of motor neurons. These findings indicate that the Optn (−/−) mice recapitulate both common and specific pathogenesis of amyotrophic lateral sclerosis associated with autophagic abnormalities. Optn (−/−) mice could serve as a mouse model for the development of therapeutic strategies.

Clinical findings and autophagic pathology in neutral lipid storage disease with myopathy.

Neutral lipid storage disease with myopathy (NLSDM) is a triglyceride metabolic disorder caused by defects of adipose triglyceride lipases (ATGL). The coexistence of lipid vacuoles and rimmed vacuoles in the myofibers is a characteristic pathological change in some NLSDM cases. However, it has not been explored whether autophagic abnormalities exist in the NLSDM myofibers with rimmed vacuole. Herein, we report that 5 patients with NLSDM initially presented with muscle weakness in the right arm related to long-term physical efforts, then developed muscle weakness of other limbs. Pathogenic mutations in the PNPLA2 gene were identified in all patients. Myopathological analysis showed a coexistence of massive lipid vacuoles and rimmed vacuoles, which was not associated with the age of onset or mutation sites, but closely related to the severity of muscle degeneration. The rimmed vacuoles showed strong immunopositivity to autophagic markers, but were negative to apoptotic markers. Significant immunoreactivity of p62 was observed in the rimmed vacuoles, while the lysosomal marker LAMP1 was severely decreased. Our study expanded the clinical and genetic spectrum of NLSDM. Loss of ATGL activity in muscle fibers with rimmed vacuoles induced a marked increase in autophagic formation, but lowered down the turnover of autolysosomes due to malfunction of lysosomes.

LC3 Immunostaining in the Inferior Olivary Nuclei of Cats With Niemann-Pick Disease Type C1 Is Associated With Patterned Purkinje Cell Loss.

The feline model of Niemann-Pick disease, type C1 (NPC1) recapitulates the clinical, neuropathological, and biochemical abnormalities present in children with NPC1. The hallmarks of disease are the lysosomal storage of unesterified cholesterol and multiple sphingolipids in neurons, and the spatial and temporal distribution of Purkinje cell death. In feline NPC1 brain, microtubule-associated protein 1 light chain 3 (LC3) accumulations, indicating autophagosomes, were found within axons and presynaptic terminals. High densities of accumulated LC3 were seen in subdivisions of the inferior olive, which project to cerebellar regions that show the most Purkinje cell loss, suggesting that autophagic abnormalities in specific climbing fibers may contribute to the spatial pattern of Purkinje cell loss seen. Biweekly intrathecal administration of 2-hydroxypropyl-beta cyclodextrin (HPβCD) ameliorated neurological dysfunction, reduced cholesterol and sphingolipid accumulation, and increased lifespan in NPC1 cats. LC3 pathology was reduced in treated animals suggesting that HPβCD administration also ameliorates autophagic abnormalities. This study is the first to (i) identify specific brain regions exhibiting autophagic abnormalities in any species with NPC1, (ii) provide evidence of differential vulnerability among discrete brain nuclei and pathways, and (iii) show the amelioration of these abnormalities in NPC1 cats treated with HPβCD.

EPG5-related Vici syndrome: a paradigm of neurodevelopmental disorders with defective autophagy.

Vici syndrome is a progressive neurodevelopmental multisystem disorder due to recessive mutations in the key autophagy gene EPG5. We report genetic, clinical, neuroradiological, and neuropathological features of 50 children from 30 families, as well as the neuronal phenotype of EPG5 knock-down in Drosophila melanogaster. We identified 39 different EPG5 mutations, most of them truncating and predicted to result in reduced EPG5 protein. Most mutations were private, but three recurrent mutations (p.Met2242Cysfs*5, p.Arg417*, and p.Gln336Arg) indicated possible founder effects. Presentation was mainly neonatal, with marked hypotonia and feeding difficulties. In addition to the five principal features (callosal agenesis, cataracts, hypopigmentation, cardiomyopathy, and immune dysfunction), we identified three equally consistent features (profound developmental delay, progressive microcephaly, and failure to thrive). The manifestation of all eight of these features has a specificity of 97%, and a sensitivity of 89% for the presence of an EPG5 mutation and will allow informed decisions about genetic testing. Clinical progression was relentless and many children died in infancy. Survival analysis demonstrated a median survival time of 24 months (95% confidence interval 0-49 months), with only a 10th of patients surviving to 5 years of age. Survival outcomes were significantly better in patients with compound heterozygous mutations (P = 0.046), as well as in patients with the recurrent p.Gln336Arg mutation. Acquired microcephaly and regression of skills in long-term survivors suggests a neurodegenerative component superimposed on the principal neurodevelopmental defect. Two-thirds of patients had a severe seizure disorder, placing EPG5 within the rapidly expanding group of genes associated with early-onset epileptic encephalopathies. Consistent neuroradiological features comprised structural abnormalities, in particular callosal agenesis and pontine hypoplasia, delayed myelination and, less frequently, thalamic signal intensity changes evolving over time. Typical muscle biopsy features included fibre size variability, central/internal nuclei, abnormal glycogen storage, presence of autophagic vacuoles and secondary mitochondrial abnormalities. Nerve biopsy performed in one case revealed subtotal absence of myelinated axons. Post-mortem examinations in three patients confirmed neurodevelopmental and neurodegenerative features and multisystem involvement. Finally, downregulation of epg5 (CG14299) in Drosophila resulted in autophagic abnormalities and progressive neurodegeneration. We conclude that EPG5-related Vici syndrome defines a novel group of neurodevelopmental disorders that should be considered in patients with suggestive features in whom mitochondrial, glycogen, or lysosomal storage disorders have been excluded. Neurological progression over time indicates an intriguing link between neurodevelopment and neurodegeneration, also supported by neurodegenerative features in epg5-deficient Drosophila, and recent implication of other autophagy regulators in late-onset neurodegenerative disease.

The update on transcriptional regulation of autophagy in normal and pathologic cells: A novel therapeutic target.

Autophagy is a genetically programmed, evolutionarily conserved catabolic process that occurs in response to stress. While short-lived proteins are degraded via the ubiquitin/proteasome pathway in higher eukaryotes, autophagy refers to intracellular pathway for development, differentiation, survival, and homeostasis through the lysosomal-dependent machinery, which regulates the synthesis, degradation, and recycling of long-lived proteins and even whole cytoplasmic organelles. The newly discovery shows that autophagy plays an important role in recycling nutrients upon starvation and maintaining cellular energy homeostasis. Recent basic and clinical investigations further demonstrate that autophagic abnormalities have been considered underlying reasons for lots of human diseases, including liver disease, cardiovascular and cerebrovascular diseases, neurodegenerative diseases, neoplastic diseases, and cancers. Pharmacological modulation of autophagy might point to possible therapeutic strategies for combating a broad range of diseases. Regrettably, although the mechanisms underlying the regulation of autophagy have long been attracted extensive concern of numerous scholars and its acute regulation by nutrient-sensing signaling pathways is well described, the overview on longer-term transcriptional regulation of autophagy is still filled with confusion. This review summarizes current knowledge on transcriptional regulation of autophagy and expands our horizons on the autophagy repertoire. On the other hand, it also offers a glimpse into different strategies that have been used in experimental models to counteract excessive pathological autophagy via transcriptional regulation mechanism in the prevention and treatment of disease.

HTT/Huntingtin in selective autophagy and Huntington disease: A foe or a friend within?

HTT/Huntingtin in selective autophagy and Huntington disease: A foe or a friend within?

The value of muscle biopsies in Pompe disease: identifying lipofuscin inclusions in juvenile- and adult-onset patients

BackgroundPompe disease, an inherited deficiency of lysosomal acid alpha-glucosidase (GAA), is a metabolic myopathy with heterogeneous clinical presentations. Late-onset Pompe disease (LOPD) is a debilitating progressive muscle disorder that can occur anytime from early childhood to late adulthood. Enzyme replacement therapy (ERT) with recombinant human GAA is currently available for Pompe patients. Although ERT shows some benefits, the reversal of skeletal muscle pathology - lysosomal glycogen accumulation and autophagic buildup - remains a challenge. In this study, we examined the clinical status and muscle pathology of 22 LOPD patients and one atypical infantile patient on ERT to understand the reasons for muscle resistance to ERT.ResultsThe patients were divided into three groups for analysis, based on the age of onset and diagnosis: adult-onset patients, juvenile-onset patients, and those identified through newborn screening (NBS). The areas of autophagic buildup found in patients’ biopsies of all three groups, contained large autofluorescent inclusions which we show are made of lipofuscin, an indigestible intralysosomal material typically associated with ageing. These inclusions, analysed by staining, spectral analysis, time-resolved Fluorescence Lifetime Imaging (FLIM), and Second Harmonic Generation (SHG) imaging, were the major pathology remaining in many fibers after ERT. The best outcome of ERT both clinically and morphologically was observed in the NBS patients.ConclusionsThe muscle biopsy, in spite of its shortcomings, allowed us to recognize an underreported, ERT-resistant pathology in LOPD; numerous lysosomes and autolysosomes loaded with lipofuscin appear to be a hallmark of LOPD skeletal muscle. Lipofuscin accumulation - a result of inefficient lysosomal degradation - may in turn exacerbate both lysosomal and autophagic abnormalities.

Cognitive Deficits and Disruption of Neurogenesis in a Mouse Model of Apolipoprotein E4 Domain Interaction

Apolipoprotein E4 (apoE4) allele is the major genetic risk factor for sporadic Alzheimer disease (AD) due to the higher prevalence and earlier onset of AD in apoE4 carriers. Accumulating data suggest that the interaction between the N- and the C-terminal domains in the protein may be the main pathologic feature of apoE4. To test this hypothesis, we used Arg-61 mice, a model of apoE4 domain interaction, by introducing the domain interaction feature of human apoE4 into native mouse apoE. We carried out hippocampus-dependent learning and memory tests and related cellular and molecular assays on 12- and 3-month-old Arg-61 and age-matched background C57BL/6J mice. Learning and memory task performance were impaired in Arg-61 mice at both old and young ages compared with C57BL/6J mice. Surprisingly, young Arg-61 mice had more mitotic doublecortin-positive cells in the subgranular zone; mRNA levels of brain-derived neurotrophic factor (BDNF) and TrkB were also higher in 3-month-old Arg-61 hippocampus compared with C57BL/6J mice. These early-age neurotrophic and neurogenic (proliferative) effects in the Arg-61 mouse may be an inadequate compensatory but eventually detrimental attempt by the system to "repair" itself. This is supported by the higher cleaved caspase-3 levels in the young animals that not only persisted, but increased in old age, and the lower levels of doublecortin at old age in the hippocampus of Arg-61 mice. These results are consistent with human apoE4-dependent cognitive and neuro-pathologic changes, supporting the principal role of domain interaction in the pathologic effect of apoE4. Domain interaction is, therefore, a viable therapeutic/prophylactic target for cognitive impairment and AD in apoE4 subjects.

Skeletal muscle pathology of infantile Pompe disease during long-term enzyme replacement therapy

BackgroundPompe disease is an autosomal recessive metabolic neuromuscular disorder caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). It has long been believed that the underlying pathology leading to tissue damage is caused by the enlargement and rupture of glycogen-filled lysosomes. Recent studies have also implicated autophagy, an intracellular lysosome-dependent degradation system, in the disease pathogenesis. In this study, we characterize the long-term impact of enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA) on lysosomal glycogen accumulation and autophagy in some of the oldest survivors with classic infantile Pompe disease (IPD).MethodsMuscle biopsies from 8 [4 female, 4 male; 6 cross-reactive immunologic material (CRIM)-positive, 2 CRIM-negative] patients with a confirmed diagnosis of classic IPD were examined using standard histopathological approaches. In addition, muscle biopsies were evaluated by immunostaining for lysosomal marker (lysosomal-associated membrane protein-2; LAMP2), autophagosomal marker (microtubule-associated protein 1 light chain 3; LC3), and acid and alkaline ATPases. All patients received rhGAA by infusion at cumulative biweekly doses of 20–40 mg/kg.ResultsMedian age at diagnosis of classic IPD was 3.4 months (range: 0 to 6.5 months; n = 8). At the time of muscle biopsy, the patients’ ages ranged from 1 to 103 months and ERT duration ranged from 0 (i.e., baseline, pre-ERT) to 96 months. The response to therapy varied considerably among the patients: some patients demonstrated motor gains while others experienced deterioration of motor function, either with or without a period of initial clinical benefit. Skeletal muscle pathology included fiber destruction, lysosomal vacuolation, and autophagic abnormalities (i.e., buildup), particularly in fibers with minimal lysosomal enlargement. Overall, the pathology reflected clinical status.ConclusionsThis is the first study to investigate the impact of rhGAA ERT on lysosomal glycogen accumulation and autophagic buildup in patients with classic IPD beyond 18 months of treatment. Our findings indicate that ERT does not fully halt or reverse the underlying skeletal muscle pathology in IPD. The best outcomes were observed in the two patients who began therapy early, namely at 0.5 and 1.1 months of age.

Dopaminergic Neuronal Loss, Reduced Neurite Complexity and Autophagic Abnormalities in Transgenic Mice Expressing G2019S Mutant LRRK2

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant familial Parkinson's disease (PD) and also contribute to idiopathic PD. LRRK2 mutations represent the most common cause of PD with clinical and neurochemical features that are largely indistinguishable from idiopathic disease. Currently, transgenic mice expressing wild-type or disease-causing mutants of LRRK2 have failed to produce overt neurodegeneration, although abnormalities in nigrostriatal dopaminergic neurotransmission have been observed. Here, we describe the development and characterization of transgenic mice expressing human LRRK2 bearing the familial PD mutations, R1441C and G2019S. Our study demonstrates that expression of G2019S mutant LRRK2 induces the degeneration of nigrostriatal pathway dopaminergic neurons in an age-dependent manner. In addition, we observe autophagic and mitochondrial abnormalities in the brains of aged G2019S LRRK2 mice and markedly reduced neurite complexity of cultured dopaminergic neurons. These new LRRK2 transgenic mice will provide important tools for understanding the mechanism(s) through which familial mutations precipitate neuronal degeneration and PD.