Increased transcription of transglutaminase 1 mediates neuronal death in in vitro models of neuronal stress and Aβ1–42-mediated toxicity

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Mutations in TGM6 induce the unfolded protein response in SCA35

Spinocerebellar ataxia type 35 (SCA35) is a rare autosomal-dominant neurodegenerative disease caused by mutations in the TGM6 gene, which codes for transglutaminase 6 (TG6). Mutations in TG6 induce cerebellar degeneration by an unknown mechanism. We identified seven patients bearing new mutations in TGM6. To gain insights into the molecular basis of mutant TG6-induced neurotoxicity, we analyzed all of the seven new TG6 mutants and the five TG6 mutants previously linked to SCA35. We found that wild-type (TG6-WT) mainly localized to the nucleus and perinuclear area, whereas five TG6 mutations showed nuclear depletion, increased accumulation in the perinuclear area, insolubility and loss of enzymatic function. Aberrant accumulation of these TG6 mutants in the perinuclear area led to activation of the unfolded protein response (UPR), suggesting that specific TG6 mutants elicit an endoplasmic reticulum (ER) stress response. Mutations associated with activation of the UPR caused death of primary neurons and reduced the survival of novel D. melanogaster models of SCA35. These results indicate that mutations differently impacting on TG6 function cause neuronal dysfunction and death through diverse mechanisms and highlight the UPR as a potential therapeutic target for patient treatment

Huntingtin-mediated axonal transport requires arginine methylation by PRMT6

The huntingtin (HTT) protein transports various organelles, including vesicles containing neurotrophic factors, from embryonic development throughout life. To better understand how HTT mediates axonal transport and why this function is disrupted in Huntington's disease (HD), we study vesicle-associated HTT and find that it is dimethylated at a highly conserved arginine residue (R118) by the protein arginine methyltransferase 6 (PRMT6). Without R118 methylation, HTT associates less with vesicles, anterograde trafficking is diminished, and neuronal death ensues—very similar to what occurs in HD. Inhibiting PRMT6 in HD cells and neurons exacerbates mutant HTT (mHTT) toxicity and impairs axonal trafficking, whereas overexpressing PRMT6 restores axonal transport and neuronal viability, except in the presence of a methylation-defective variant of mHTT. In HD flies, overexpressing PRMT6 rescues axonal defects and eclosion. Arginine methylation thus regulates HTT-mediated vesicular transport along the axon, and increasing HTT methylation could be of therapeutic interest for HD.Telethon-Italy and Autonomous Province of Trento (TCP12013 to M.P.); Association Française contre les Myopathies (AFM-22221 to M.P. and M.B.); PRIN-MUR (2017F2A2C5 to M.P.); National Institutes of Health (1R21NS111768-01 to M.P. and U.B.P.); PROGRAM RARE DISEASES CNCCS-Scarl-Pomezia (M.P.); FONDAZIONE AIRC-Italy (24423 to M.P.); Alzheimer Trento Onlus with the legato Baldrachi (M.B.); the Agence Nationale de la Recherche (ANR-15-JPWG-0003-05 JPND CIRCPROT and ANR-18-CE16-0009-01 AXYON to F.S.) and the Spanish Ministry of Science, Innovation and Universities (RTI2018-096322-B-I00 MCIU/AEI/FEDER-UE to J.J.L.

Transplantation studies in animal models of Parkinson�'s Disease

Parkinson’s disease (PD) is a neurodegenerative disorder, resulting from the loss of dopaminergic (DA-ergic) neurons from the substantia nigra pars compacta (SNpc) region of the brain. These cells form an integral part of the basal ganglia, an area responsible for motor coordination in mammals. In humans and subhuman primates, loss of these cells results in akinesia, rigidity, postural instability and tremor, and these symptoms progress as the loss of neurons from this region accelerates. Cell replacement therapy is considered as an obvious option for the recovery of lost motor functions resulting from the loss of neurons. This is relevant for PD owing to failures of available pharmacological approaches. Moreover, it was thought to be theoretically feasible because of the loss of only a particular segment of the CNS DA-ergic systems, the A9 DA-ergic neurons, the terminals of which are in each of the striatum. Additionally, the anatomical location of the terminal region is an accessible site for any surgical interventions. A simple technique of placing dopamine secreting cells at the site of the A9 SNpc neuronal terminals would provide the neurotransmitter required for stimulating postsynaptic neurons in the area. The next problem could be engineering the transplanted neurons to release dopamine to specific stimuli. However, in practice, even transplanting dopamine releasing neurons in the striatum met with difficulties. To add to the woes, the varied outcomes of transplantation studies made it difficult to decipher the exact problem(s). Ultimately, what remains as of now are a bunch of speculations in regard to the failures or inconclusive outcomes of this therapeutic option. The major problems associated with cell replacement therapy in PD seem to be (i) the availability of implant material, (ii) its source, (iii) nature of the material, (iv) survivability of the cells in the grafts, and finally (v) fate of the transplants in long term grafts. Last, but not the least, the graft associated abnormalities in the host is a major unresolved issue

Engraftment of Mouse Embryonic Stem Cells Differentiated by Default Leads to Neuroprotection, Behaviour Revival and Astrogliosis in Parkinsonian Rats

<div><p>We report here protection against rotenone-induced behavioural dysfunction, striatal dopamine depletion and nigral neuronal loss, following intra-striatal transplantation of neurons differentiated from murine embryonic stem cells (mES). mES maintained in serum free medium exhibited increase in neuronal, and decrease in stem cell markers by 7th and 10th days as revealed by RT-PCR and immunoblot analyses. Tyrosine hydroxylase, NURR1, PITX3, LMX1b and c-RET mRNA showed a significant higher expression in differentiated cells than in mES. Dopamine level was increased by 3-fold on 10th day as compared to 7 days differentiated cells. Severity of rotenone-induced striatal dopamine loss was attenuated, and amphetamine-induced unilateral rotations were significantly reduced in animals transplanted with 7 days differentiated cells, but not in animals that received undifferentiated ES transplant. However, the ratio of contralateral to ipsilateral swings in elevated body swing test was significantly reduced in both the transplanted groups, as compared to control. Striatal grafts exhibited the presence of tyrosine hydroxylase positive cells, and the percentage of dopaminergic neurons in the substantia nigra was also found to be higher in the ipsilateral side of 7 days and mES grafted animals. Increased expression of CD11b and IBA-1, suggested a significant contribution of these microglia-derived factors in controlling the limited survival of the grafted cells. Astrocytosis in the grafted striatum, and significant increase in the levels of glial cell line derived neurotrophic factor may have contributed to the recovery observed in the hemiparkinsonian rats following transplantation.</p></div

Transplantation-induced functional recovery.

<p>Bar graphs in (A) and (B) depict the number of unilateral rotations in hemiparkinsonian rats infused with vehicle only without any cells (ROT), or transplanted with 5×10⁵ embryonic stem cells, undifferentiated (ES) or 7 days differentiated cells (7 d) in the striatum, and treated with amphetamine (Amp) or apomorphine (Apo) respectively on 14th day or 16th days post-transplantation. Amphetamine caused ipsilateral rotations, whereas apomorphine caused contralateral rotations. (C) Preferential bias in body swings, recorded on the 47th day post-lesion or 17th day post transplantation. *<i>p</i>≤0.05 as compared to the pre-transplanted values of rotations, which was similar to vehicle infused group, n = 6–7. (D) Striatal DA levels in the ROT, ES and 7 d groups. *<i>p</i>≤0.05 as compared to the control contralateral side, ^@<i>p</i>≤0.05 as compared to ES transplanted striatum, n = 5–6.</p

Neurons in the nigra of the animals that received unilaterally grafts in the striatum.

<p>Sections passing through the substantia nigra pars compacta were cut and immunostained for tyrosine hydroxylase (arrows; ipsilateral nigra) (A–C) or stained for neurons with cresyl violet (D–F). The sections passing through nigral region of the midbrain of rats that received the vehicle, without cells (A, D), undifferentiated embryonic stem cells (ES; B,E) or 7 days differentiated ES (7 d; C,F). There appeared a significant percentage of improvement in the total number of neurons in the ipsilateral substantia nigra relative to the contralateral side (G), and in TH-positive dopaminergic neurons (H). Results are presented as Mean ± SEM. *<i>p</i>≤0.05, as compared to control or ES transplanted group. Sections from 3 different brain samples were considered for each group.</p

Presence of serotonin and GFAP positive cells in the differentiated cells.

<p>Immunocytochemical localization of serotonin (5-HT; B,C) and glial fibrillary acidic protein (GFAP; E,F) in 7 days differentiated cells showing glial (E) and few serotoninergic neurons (B) in the culture. Scale bar 10 µm.</p