Identification and characterization of novel ERBB4 variant associated with sporadic amyotrophic lateral sclerosis (ALS).

Amyotrophic Lateral Sclerosis (ALS) is the most common type of motor neuron disease characterized by progressive motor neuron degeneration in brain and spinal cord. Most cases are sporadic in ALS and 5-10% of cases are familiar. >50 genes are known to be associated with ALS and one of them is ERBB4. In this paper, we report the case of a 53-year-old ALS patient with progressive muscle weakness and fasciculation, but he had no cognitive decline. We performed the next generation sequencing (NGS) and in silico analysis, it predicted a highly pathogenic variant, c.2116 A > G, p.Asn706Asp (N706D) in the ERBB4 gene. The amino acid residue is highly conserved among species. ERBB4 is a member of the ERBB family of receptor tyrosine kinases. ERBB4 has multiple tyrosine phosphorylation sites, including an autophosphorylation site at tyrosine 1284 residue. Autophosphorylation of ERBB4 promotes biological activity and it associated with NRG-1/ERBB4 pathway. It is already known that tyrosine 128 phosphorylation of ERBB4 is decreased in patients who have ALS-associated ERBB4 mutations. We generated ERBB4 N706D construct using site-directed mutagenesis and checked the phosphorylation level of ERBB4 N706D in NSC-34 cells. We found that the phosphorylation of ERBB4 N706D was decreased compared to ERBB4 wild-type, indicating a loss of function mutation in ERBB4. We report a novel variant in ERBB4 gene leading to ALS through dysfunction of ERBB4.

Potential roles of the endoplasmic reticulum stress pathway in amyotrophic lateral sclerosis.

The endoplasmic reticulum (ER) is a major organelle involved in protein quality control and cellular homeostasis. ER stress results from structural and functional dysfunction of the organelle, along with the accumulation of misfolded proteins and changes in calcium homeostasis, it leads to ER stress response pathway such as unfolded protein response (UPR). Neurons are particularly sensitive to the accumulation of misfolded proteins. Thus, the ER stress is involved in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, prion disease and motor neuron disease (MND). Recently, the complex involvement of ER stress pathways has been demonstrated in experimental models of amyotrophic lateral sclerosis (ALS)/MND using pharmacological and genetic manipulation of the unfolded protein response (UPR), an adaptive response to ER stress. Here, we aim to provide recent evidence demonstrating that the ER stress pathway is an essential pathological mechanism of ALS. In addition, we also provide therapeutic strategies that can help treat diseases by targeting the ER stress pathway.

PTK2 regulates tau-induced neurotoxicity via phosphorylation of p62 at Ser403.

Tau is a microtubule-associated protein that forms insoluble filaments that accumulate as neurofibrillary tangles in neurodegenerative diseases such as Alzheimer's disease and other related tauopathies. A relationship between abnormal Tau accumulation and ubiquitin-proteasome system impairment has been reported. However, the molecular mechanism linking Tau accumulation and ubiquitin proteasome system (UPS) dysfunction remains unclear. Here, we show that overexpression of wild-type or mutant (P301L) Tau increases the abundance of polyubiquitinated proteins and activates the autophagy-lysosome pathway in mammalian neuronal cells. Previous studies found that PTK2 inhibition mitigates toxicity induced by UPS impairment. Thus, we investigated whether PTK2 inhibition can attenuate Tau-induced UPS impairment and cell toxicity. We found that PTK2 inhibition significantly reduces Tau-induced death in mammalian neuronal cells. Moreover, overexpression of WT or mutant Tau increased the phosphorylation levels of PTK2 and p62. We also confirmed that PTK2 inhibition suppresses Tau-induced phosphorylation of PTK2 and p62. Furthermore, PTK2 inhibition significantly attenuated the climbing defect and shortened the lifespan in the Drosophila model of tauopathy. In addition, we observed that phosphorylation of p62 is markedly increased in Alzheimer's disease patients with tauopathies. Taken together, our results indicate that the UPS dysfunction induced by Tau accumulation might contribute directly to neurodegeneration in tauopathies and that PTK2 could be a promising therapeutic target for tauopathies.

HEXA-018, a Novel Inducer of Autophagy, Rescues TDP-43 Toxicity in Neuronal Cells.

The autophagy-lysosomal pathway is an essential cellular mechanism that degrades aggregated proteins and damaged cellular components to maintain cellular homeostasis. Here, we identified HEXA-018, a novel compound containing a catechol derivative structure, as a novel inducer of autophagy. HEXA-018 increased the LC3-I/II ratio, which indicates activation of autophagy. Consistent with this result, HEXA-018 effectively increased the numbers of autophagosomes and autolysosomes in neuronal cells. We also found that the activation of autophagy by HEXA-018 is mediated by the AMPK-ULK1 pathway in an mTOR-independent manner. We further showed that ubiquitin proteasome system impairment- or oxidative stress-induced neurotoxicity was significantly reduced by HEXA-018 treatment. Moreover, oxidative stress-induced mitochondrial dysfunction was strongly ameliorated by HEXA-018 treatment. In addition, we investigated the efficacy of HEXA-018 in models of TDP-43 proteinopathy. HEXA-018 treatment mitigated TDP-43 toxicity in cultured neuronal cell lines and Drosophila. Our data indicate that HEXA-018 could be a new drug candidate for TDP-43-associated neurodegenerative diseases.

Vitamin B12 Reduces TDP-43 Toxicity by Alleviating Oxidative Stress and Mitochondrial Dysfunction.

TAR DNA-binding protein 43 (TDP-43) is a member of an evolutionarily conserved family of heterogeneous nuclear ribonucleoproteins that modulate multiple steps in RNA metabolic processes. Cytoplasmic aggregation of TDP-43 in affected neurons is a pathological hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer's disease (AD), and limbic predominant age-related TDP-43 encephalopathy (LATE). Mislocalized and accumulated TDP-43 in the cytoplasm induces mitochondrial dysfunction and reactive oxidative species (ROS) production. Here, we show that TDP-43- and rotenone-induced neurotoxicity in the human neuronal cell line SH-SY5Y were attenuated by hydroxocobalamin (Hb, vitamin B12 analog) treatment. Although Hb did not affect the cytoplasmic accumulation of TDP-43, Hb attenuated TDP-43-induced toxicity by reducing oxidative stress and mitochondrial dysfunction. Moreover, a shortened lifespan and motility defects in TDP-43-expressing Drosophila were significantly mitigated by dietary treatment with hydroxocobalamin. Taken together, these findings suggest that oral intake of hydroxocobalamin may be a potential therapeutic intervention for TDP-43-associated proteinopathies.

The Role of HDAC6 in TDP-43-Induced Neurotoxicity and UPS Impairment.

Transactive response DNA-binding protein 43 (TDP-43)-induced neurotoxicity is currently well recognized as a contributor to the pathology of amyotrophic lateral sclerosis (ALS), and the deposition of TDP-43 has been linked to other neurodegenerative diseases, such as frontotemporal lobar degeneration (FTLD) and Alzheimer's disease (AD). Recent studies also suggest that TDP-43-induced neurotoxicity is associated with ubiquitin-proteasome system (UPS) impairment. Histone deacetylase 6 (HDAC6) is a well-known cytosolic deacetylase enzyme that suppresses the toxicity of UPS impairment. However, the role of HDAC6 in TDP-43-induced neurodegeneration is largely unknown. In this study, we found that HDAC6 overexpression decreased the levels of insoluble and cytosolic TDP-43 protein in TDP-43-overexpressing N2a cells. In addition, TDP-43 overexpression upregulated HDAC6 protein and mRNA levels, and knockdown of Hdac6 elevated the total protein level of TDP-43. We further found that HDAC6 modulates TDP-43-induced UPS impairment via the autophagy-lysosome pathway (ALP). We also showed that TDP-43 promoted a short lifespan in flies and that the accumulation of ubiquitin aggregates and climbing defects were significantly rescued by overexpression of HDAC6 in flies. Taken together, these findings suggest that HDAC6 overexpression can mitigate neuronal toxicity caused by TDP-43-induced UPS impairment, which may represent a novel therapeutic approach for ALS.

The Role of Glial Mitochondria in α-Synuclein Toxicity.

The abnormal accumulation of alpha-synuclein (α-syn) aggregates in neurons and glial cells is widely known to be associated with many neurodegenerative diseases, including Parkinson's disease (PD), Dementia with Lewy bodies (DLB), and Multiple system atrophy (MSA). Mitochondrial dysfunction in neurons and glia is known as a key feature of α-syn toxicity. Studies aimed at understanding α-syn-induced toxicity and its role in neurodegenerative diseases have primarily focused on neurons. However, a growing body of evidence demonstrates that glial cells such as microglia and astrocytes have been implicated in the initial pathogenesis and the progression of α-Synucleinopathy. Glial cells are important for supporting neuronal survival, synaptic functions, and local immunity. Furthermore, recent studies highlight the role of mitochondrial metabolism in the normal function of glial cells. In this work, we review the complex relationship between glial mitochondria and α-syn-mediated neurodegeneration, which may provide novel insights into the roles of glial cells in α-syn-associated neurodegenerative diseases.

The overexpression of TDP-43 in astrocytes causes neurodegeneration via a PTP1B-mediated inflammatory response.

BACKGROUND: Cytoplasmic inclusions of transactive response DNA binding protein of 43 kDa (TDP-43) in neurons and astrocytes are a feature of some neurodegenerative diseases, such as frontotemporal lobar degeneration with TDP-43 (FTLD-TDP) and amyotrophic lateral sclerosis (ALS). However, the role of TDP-43 in astrocyte pathology remains largely unknown. METHODS: To investigate whether TDP-43 overexpression in primary astrocytes could induce inflammation, we transfected primary astrocytes with plasmids encoding Gfp or TDP-43-Gfp. The inflammatory response and upregulation of PTP1B in transfected cells were examined using quantitative RT-PCR and immunoblot analysis. Neurotoxicity was analysed in a transwell coculture system of primary cortical neurons with astrocytes and cultured neurons treated with astrocyte-conditioned medium (ACM). We also examined the lifespan, performed climbing assays and analysed immunohistochemical data in pan-glial TDP-43-expressing flies in the presence or absence of a Ptp61f RNAi transgene. RESULTS: PTP1B inhibition suppressed TDP-43-induced secretion of inflammatory cytokines (interleukin 1 beta (IL-1ß), interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α)) in primary astrocytes. Using a neuron-astrocyte coculture system and astrocyte-conditioned media treatment, we demonstrated that PTP1B inhibition attenuated neuronal death and mitochondrial dysfunction caused by overexpression of TDP-43 in astrocytes. In addition, neuromuscular junction (NMJ) defects, a shortened lifespan, inflammation and climbing defects caused by pan-glial overexpression of TDP-43 were significantly rescued by downregulation of ptp61f (the Drosophila homologue of PTP1B) in flies. CONCLUSIONS: These results indicate that PTP1B inhibition mitigates the neuronal toxicity caused by TDP-43-induced inflammation in mammalian astrocytes and Drosophila glial cells.

The role of TDP-43 propagation in neurodegenerative diseases: integrating insights from clinical and experimental studies.

TAR DNA-binding protein 43 (TDP-43) is a highly conserved nuclear RNA/DNA-binding protein involved in the regulation of RNA processing. The accumulation of TDP-43 aggregates in the central nervous system is a common feature of many neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer's disease (AD), and limbic predominant age-related TDP-43 encephalopathy (LATE). Accumulating evidence suggests that prion-like spreading of aberrant protein aggregates composed of tau, amyloid-ß, and α-synuclein is involved in the progression of neurodegenerative diseases such as AD and PD. Similar to those of prion-like proteins, pathological aggregates of TDP-43 can be transferred from cell-to-cell in a seed-dependent and self-templating manner. Here, we review clinical and experimental studies supporting the prion-like spreading of misfolded TDP-43 and discuss the molecular mechanisms underlying the propagation of these pathological aggregated proteins. The idea that misfolded TDP-43 spreads in a prion-like manner between cells may guide novel therapeutic strategies for TDP-43-associated neurodegenerative diseases.

PTK2/FAK regulates UPS impairment via SQSTM1/p62 phosphorylation in TARDBP/TDP-43 proteinopathies.

TARDBP/TDP-43 (TAR DNA binding protein) proteinopathies are a common feature in a variety of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and Alzheimer disease (AD). However, the molecular mechanisms underlying TARDBP-induced neurotoxicity are largely unknown. In this study, we demonstrated that TARDBP proteinopathies induce impairment in the ubiquitin proteasome system (UPS), as evidenced by an accumulation of ubiquitinated proteins and a reduction in proteasome activity in neuronal cells. Through kinase inhibitor screening, we identified PTK2/FAK (PTK2 protein tyrosine kinase 2) as a suppressor of neurotoxicity induced by UPS impairment. Importantly, PTK2 inhibition significantly reduced ubiquitin aggregates and attenuated TARDBP-induced cytotoxicity in a Drosophila model of TARDBP proteinopathies. We further identified that phosphorylation of SQSTM1/p62 (sequestosome 1) at S403 (p-SQSTM1 [S403]), a key component in the autophagic degradation of poly-ubiquitinated proteins, is increased upon TARDBP overexpression and is dependent on the activation of PTK2 in neuronal cells. Moreover, expressing a non-phosphorylated form of SQSTM1 (SQSTM1S403A) significantly repressed the accumulation of insoluble poly-ubiquitinated proteins and neurotoxicity induced by TARDBP overexpression in neuronal cells. In addition, TBK1 (TANK binding kinase 1), a kinase that phosphorylates S403 of SQSTM1, was found to be involved in the PTK2-mediated phosphorylation of SQSTM1. Taken together, our data suggest that the PTK2-TBK1-SQSTM1 axis plays a critical role in the pathogenesis of TARDBP by regulating neurotoxicity induced by UPS impairment. Therefore, targeting the PTK2-TBK1-SQSTM1 axis may represent a novel therapeutic intervention for neurodegenerative diseases with TARDBP proteinopathies.Abbreviations: ALP: macroautophagy/autophagy lysosomal pathway; ALS: amyotrophic lateral sclerosis; ATXN2: ataxin 2; BafA1: bafilomycin A1; cCASP3: cleaved caspase 3; CSNK2: casein kinase 2; FTLD: frontotemporal lobar degeneration; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; OPTN: optineurin; PTK2/FAK: PTK2 protein tyrosine kinase 2; SQSTM1/p62: sequestosome 1; TARDBP/TDP-43: TAR DNA binding protein; TBK1: TANK binding kinase 1; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system.

Neuroprotective Effects of Protein Tyrosine Phosphatase 1B Inhibition against ER Stress-Induced Toxicity.

Several lines of evidence suggest that endoplasmic reticulum (ER) stress plays a critical role in the pathogenesis of many neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Protein tyrosine phosphatase 1B (PTP1B) is known to regulate the ER stress signaling pathway, but its role in neuronal systems in terms of ER stress remains largely unknown. Here, we showed that rotenone-induced toxicity in human neuroblastoma cell lines and mouse primary cortical neurons was ameliorated by PTP1B inhibition. Moreover, the increase in the level of ER stress markers (eIF2α phosphorylation and PERK phosphorylation) induced by rotenone treatment was obviously suppressed by concomitant PTP1B inhibition. However, the rotenone-induced production of reactive oxygen species (ROS) was not affected by PTP1B inhibition, suggesting that the neuroprotective effect of the PTP1B inhibitor is not associated with ROS production. Moreover, we found that MG132-induced toxicity involving proteasome inhibition was also ameliorated by PTP1B inhibition in a human neuroblastoma cell line and mouse primary cortical neurons. Consistently, downregulation of the PTP1B homologue gene in Drosophila mitigated rotenone- and MG132-induced toxicity. Taken together, these findings indicate that PTP1B inhibition may represent a novel therapeutic approach for ER stress-mediated neurodegenerative diseases.