The hereditary spastic paraplegias (HSPs) are a heterogeneous group of motorneuron diseases characterized by progressive spasticity and paresis of the lower limbs. Mutations in Spastic Gait 4 (SPG4), encoding spastin, are the most frequent cause of HSP. To understand how mutations in SPG4 affect human neurons, we generated human induced pluripotent stem cells (hiPSCs) from fibroblasts of two patients carrying a c.1684C>T nonsense mutation and from two controls. These SPG4 and control hiPSCs were able to differentiate into neurons and glia at comparable efficiency. All known spastin isoforms were reduced in SPG4 neuronal cells. The complexity of SPG4 neurites was decreased, which was paralleled by an imbalance of axonal transport with less retrograde movement. Prominent neurite swellings with disrupted microtubules were present in SPG4 neurons at an ultrastructural level. While some of these swellings contain acetylated and detyrosinated tubulin, these tubulin modifications were unchanged in total cell lysates of SPG4 neurons. Upregulation of another microtubule-severing protein, p60 katanin, may partially compensate for microtubuli dynamics in SPG4 neurons. Overexpression of the M1 or M87 spastin isoforms restored neurite length, branching, numbers of primary neurites and reduced swellings in SPG4 neuronal cells. We conclude that neurite complexity and maintenance in HSP patient-derived neurons are critically sensitive to spastin gene dosage. Our data show that elevation of single spastin isoform levels is sufficient to restore neurite complexity and reduce neurite swellings in patient cells. Furthermore, our human model offers an ideal platform for pharmacological screenings with the goal to restore physiological spastin levels in SPG4 patients.

Hereditary spastic paraplegias are a group of inherited motor neuron diseases characterized by progressive paraparesis and spasticity. Mutations in the spastic paraplegia gene SPG11, encoding spatacsin, cause an autosomal-recessive disease trait; however, the precise knowledge about the role of spatacsin in neurons is very limited. We for the first time analyzed the expression and function of spatacsin in human forebrain neurons derived from human pluripotent stem cells including lines from two SPG11 patients and two controls. SPG11 patients'-derived neurons exhibited downregulation of specific axonal-related genes, decreased neurite complexity and accumulation of membranous bodies within axonal processes. Altogether, these data point towards axonal pathologies in human neurons with SPG11 mutations. To further corroborate spatacsin function, we investigated human pluripotent stem cell-derived neurons and mouse cortical neurons. In these cells, spatacsin was located in axons and dendrites. It colocalized with cytoskeletal and synaptic vesicle (SV) markers and was present in synaptosomes. Knockdown of spatacsin in mouse cortical neurons evidenced that the loss of function of spatacsin leads to axonal instability by downregulation of acetylated tubulin. Finally, time-lapse assays performed in SPG11 patients'-derived neurons and spatacsin-silenced mouse neurons highlighted a reduction in the anterograde vesicle trafficking indicative of impaired axonal transport. By employing SPG11 patient-derived forebrain neurons and mouse cortical neurons, this study provides the first evidence that SPG11 is implicated in axonal maintenance and cargo trafficking. Understanding the cellular functions of spatacsin will allow deciphering mechanisms of motor cortex dysfunction in autosomal-recessive hereditary spastic paraplegia.

ObjectiveMutations in the spastic paraplegia gene 11 (SPG11), encoding spatacsin, cause the most frequent form of autosomal‐recessive complex hereditary spastic paraplegia (HSP) and juvenile‐onset amyotrophic lateral sclerosis (ALS5). When SPG11 is mutated, patients frequently present with spastic paraparesis, a thin corpus callosum, and cognitive impairment. We previously delineated a neurodegenerative phenotype in neurons of these patients. In the current study, we recapitulated early developmental phenotypes of SPG11 and outlined their cellular and molecular mechanisms in patient‐specific induced pluripotent stem cell (iPSC)‐derived cortical neural progenitor cells (NPCs).MethodsWe generated and characterized iPSC‐derived NPCs and neurons from 3 SPG11 patients and 2 age‐matched controls.ResultsGene expression profiling of SPG11‐NPCs revealed widespread transcriptional alterations in neurodevelopmental pathways. These include changes in cell‐cycle, neurogenesis, cortical development pathways, in addition to autophagic deficits. More important, the GSK3ß‐signaling pathway was found to be dysregulated in SPG11‐NPCs. Impaired proliferation of SPG11‐NPCs resulted in a significant diminution in the number of neural cells. The decrease in mitotically active SPG11‐NPCs was rescued by GSK3 modulation.InterpretationThis iPSC‐derived NPC model provides the first evidence for an early neurodevelopmental phenotype in SPG11, with GSK3ß as a potential novel target to reverse the disease phenotype. Ann Neurol 2016;79:826–840

Bipolar disorder (BD) is a neuropsychiatric illness defined by recurrent episodes of mania/hypomania, depression and circadian rhythm abnormalities. Lithium is an effective drug for BD, but 30–40% of patients fail to respond adequately to treatment. Previous work has demonstrated that lithium affects the expression of “clock genes” and that lithium responders (Li-R) can be distinguished from non-responders (Li-NR) by differences in circadian rhythms. However, circadian rhythms have not been evaluated in BD patient neurons from Li-R and Li-NR. We used induced pluripotent stem cells (iPSCs) to culture neuronal precursor cells (NPC) and glutamatergic neurons from BD patients characterized for lithium responsiveness and matched controls. We identified strong circadian rhythms in Per2-luc expression in NPCs and neurons from controls and Li-R, but NPC rhythms in Li-R had a shorter circadian period. Li-NR rhythms were low-amplitude and profoundly weakened. In NPCs and neurons, expression of PER2 was higher in both BD groups compared to controls. In neurons, PER2 protein levels were higher in BD than controls, especially in Li-NR samples. In single cells, NPC and neuron rhythms in both BD groups were desynchronized compared to controls. Lithium lengthened period in Li-R and control neurons but failed to alter rhythms in Li-NR. In contrast, temperature entrainment increased amplitude across all groups, and partly restored rhythms in Li-NR neurons. We conclude that neuronal circadian rhythm abnormalities are present in BD and most pronounced in Li-NR. Rhythm deficits in BD may be partly reversible through stimulation of entrainment pathways.

Spastic paraplegia gene 11(SPG11)-linked hereditary spastic paraplegia is a complex monogenic neurodegenerative disease that in addition to spastic paraplegia is characterized by childhood onset cognitive impairment, thin corpus callosum and enlarged ventricles. We have previously shown impaired proliferation of SPG11 neural progenitor cells (NPCs). For the delineation of potential defect in SPG11 brain development we employ 2D culture systems and 3D human brain organoids derived from SPG11 patients’ iPSC and controls. We reveal that an increased rate of asymmetric divisions of NPCs leads to proliferation defect, causing premature neurogenesis. Correspondingly, SPG11 organoids appeared smaller than controls and had larger ventricles as well as thinner germinal wall. Premature neurogenesis and organoid size were rescued by GSK3 inhibititors including the Food and Drug Administration-approved tideglusib. These findings shed light on the neurodevelopmental mechanisms underlying disease pathology.

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TOR (target of rapamycin) protein kinase acts as a central controller of cell growth and development of an organism. Present study was undertaken to find the expression pattern and role of TOR during growth and development of Dictyostelium discoideum. Failures to generate either knockout and/or knockdown mutants indicate that interference with its levels led to cellular defects. Thus, the effects of TOR (DDB_G0281569) overexpression specifically, cells expressing Dd(Δ211-TOR)-Eyfp mutant was analyzed. Elevated expression of (Δ211-TOR)-Eyfp reduced both cell size and cell proliferation. DdTOR was found to be closer to fungus. mRNA level of TOR was found maximally in the freshly starved/aggregate cells that gradually declined. This was also strengthened by the expression patterns observed by in situ and the analysis of β-galactosidase reporter driven by the putative TOR promoter. The TOR protein was found to be highest at the aggregate stage. The fusion protein, (Δ211-TOR)-Eyfp was localized to the cell membrane, cytosol, and the nucleus. We suggest, DdTOR to be an essential protein and high TOR expression inhibits cell proliferation.

BackgroundTrophic factors (TFs) play important role during development and adult tissue maintenance. In neurodegenerative diseases (ND) TF supplementation provides protection. Stromal cells (HUMS) derived from the human umbilical cord matrix provide neuroprotection in the ND models of mice.PurposeThough TF mediated protection is known, the exact mechanism of protection is not clear. So, here the essential TFs (secreted by HUMS cells) and the pathway of induction of neurite extension, differentiation and networking is addressed.MethodsThe HUMS cells from the human umbilical cord matrix were derived and the mouse spinal cord motor neuron cell line, NSC-34 was extensively used. Flow cytometry, immunohistochemistry, RT- PCR, western blot, ELISA and antibody/inhibitor treatment were carried out to figure out the TF pathway.ResultsThe HUMS cells secrete six neurotrophic factors (sTFs), namely, NT-3, NGF, BDNF, VEGF, IGF-1 and GDNF (TFs). These TFs are sufficient to induce differentiation, neurite extension and neural networking in a motor neuron cell line, NSC34. All the 5 TFs need to be neutralized simultaneously with their antibodies to abrogate neurite extension. These motor neurons express the concomitant receptors, which are either receptor tyrosine kinase (TrK) coupled or to the receptor followed by the TrKs, for the above trophic factors (except for BDNF). The tyrosine kinase inhibitor, K252a, drastically reduces neurite extension. In NSC34, the TFs are coupled to the PI3K–Akt–pathway and the RAS-MAP kinase signaling through phosphorylation of ERK1 and ERK2. PI3K inhibitor, Ly 294002, abolishes neural differentiation and neurite extension. Thus, differentiation, neurite extension and networking could be achieved through the PI3K pathway. Intriguingly, the cAMP second messenger system coupling was not required. H89, PKA-inhibitor caused extensive cell death. But, had no effect in the presence of HUMS-secreted-TFs(HSTFs) suggesting a pathway switch for cell survival itself.ConclusionHUMS cells and their secreted factors could be of great use in regenerative medicine (RM). The activators of PI3K pathway, the major route of these HUMS-TFs action could be explored in RM and in the neurobiology of neural differentiation and extension.