SUMO modifies GßL and mediates mTOR signaling.

The mechanistic target of rapamycin (mTOR) signaling is influenced by multiple regulatory proteins and post-translational modifications; however, underlying mechanisms remain unclear. Here, we report a novel role of small ubiquitin-like modifier (SUMO) in mTOR complex assembly and activity. By investigating the SUMOylation status of core mTOR components, we observed that the regulatory subunit, GßL (G protein ß-subunit-like protein, also known as mLST8), is modified by SUMO1, 2, and 3 isoforms. Using mutagenesis and mass spectrometry, we identified that GßL is SUMOylated at lysine sites K86, K215, K245, K261, and K305. We found that SUMO depletion reduces mTOR-Raptor (regulatory protein associated with mTOR) and mTOR-Rictor (rapamycin-insensitive companion of mTOR) complex formation and diminishes nutrient-induced mTOR signaling. Reconstitution with WT GßL but not SUMOylation-defective KR mutant GßL promotes mTOR signaling in GßL-depleted cells. Taken together, we report for the very first time that SUMO modifies GßL, influences the assembly of mTOR protein complexes, and regulates mTOR activity.

Characterization of Mitochondria Degeneration in Spinal Motor Neurons Triggered by Chronic Over-activation of α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid Receptors in the Rat Spinal Cord in Vivo.

Mitochondrial damage is a central mechanism involved in neurological disorders as Alzheimer's, and Parkinson's diseases and amyotrophic lateral sclerosis. Energy production is the most studied mitochondrial function; however, mitochondria are also involved in processes like calcium buffering homeostasis, and cell death control during apoptosis and necrosis. Using transmission electron microscopy, in this in vivo study in male rats, we describe ultrastructural mitochondrial alterations of spinal motor neurons along chronic AMPA-induced excitotoxicity, which has been described as one of the most relevant mechanisms in ALS disease. Mitochondrial alterations begin with a crest swelling, which progresses to a full mitochondrial swelling and crest disruption. Changes on the mitochondrial morphology from elongated to a circular shape also occur along the AMPA-excitotoxicity process. In addition, by combining the TUNEL assay and immunohistochemistry for mitochondrial enzymes, we show evidence of mitochondrial DNA damage. Evidence of mitochondrial alterations during an AMPA-excitotoxic event is relevant because resembles the mitochondrial alterations previously reported in ALS patients and in transgenic familial ALS models, suggesting that a chronic excitotoxic model can be related to sporadic ALS (as has been shown in recent papers), which represent more than the 90% of the ALS cases. Understanding the mechanisms involved in motor neuron degenerative process, such as the ultrastructural mitochondrial changes permits to design strategies for MN-degeneration treatments in ALS.

Decoding the Therapeutic Implications of the ERα Stability and Subcellular Distribution in Breast Cancer.

Approximately 70% of all breast cancer cases are estrogen receptor-alpha positive (ERα+) and any ERα signaling pathways deregulation is critical for the progression of malignant mammary neoplasia. ERα acts as a transcription factor that promotes the expression of estrogen target genes associated with pro-tumor activity in breast cancer cells. Furthermore, ERα is also part of extranuclear signaling pathways related to endocrine resistance. The regulation of ERα subcellular distribution and protein stability is critical to regulate its functions and, consequently, influence the response to endocrine therapies and progression of this pathology. This minireview highlights studies that have deciphered the molecular mechanisms implicated in controlling ERα stability and nucleo-cytoplasmic transport. These mechanisms offer information about novel biomarkers, therapeutic targets, and promising strategies for breast cancer treatment.

Rhes protein transits from neuron to neuron and facilitates mutant huntingtin spreading in the brain.

Rhes (RASD2) is a thyroid hormone-induced gene that regulates striatal motor activity and promotes neurodegeneration in Huntington disease (HD) and tauopathy. Rhes moves and transports the HD protein, polyglutamine-expanded huntingtin (mHTT), via tunneling nanotube (TNT)-like membranous protrusions between cultured neurons. However, similar intercellular Rhes transportation in the intact brain was unknown. Here, we report that Rhes induces TNT-like protrusions in the striatal medium spiny neurons (MSNs) and transported between dopamine-1 receptor (D1R)-MSNs and D2R-MSNs of intact striatum and organotypic brain slices. Notably, mHTT is robustly transported within the striatum and from the striatum to the cortical areas in the brain, and Rhes deletion diminishes such transport. Moreover, Rhes moves to the cortical regions following restricted expression in the MSNs of the striatum. Thus, Rhes is a first striatum-enriched protein demonstrated to move and transport mHTT between neurons and brain regions, providing new insights into interneuronal protein transport in the brain.

Deletion of SUMO1 attenuates behavioral and anatomical deficits by regulating autophagic activities in Huntington disease.

The CAG expansion of huntingtin (mHTT) associated with Huntington disease (HD) is a ubiquitously expressed gene, yet it prominently damages the striatum and cortex, followed by widespread peripheral defects as the disease progresses. However, the underlying mechanisms of neuronal vulnerability are unclear. Previous studies have shown that SUMO1 (small ubiquitin-like modifier-1) modification of mHtt promotes cellular toxicity, but the in vivo role and functions of SUMO1 in HD pathogenesis are unclear. Here, we report that SUMO1 deletion in Q175DN HD-het knockin mice (HD mice) prevented age-dependent HD-like motor and neurological impairments and suppressed the striatal atrophy and inflammatory response. SUMO1 deletion caused a drastic reduction in soluble mHtt levels and nuclear and extracellular mHtt inclusions while increasing cytoplasmic mHtt inclusions in the striatum of HD mice. SUMO1 deletion promoted autophagic activity, characterized by augmented interactions between mHtt inclusions and a lysosomal marker (LAMP1), increased LC3B- and LAMP1 interaction, and decreased interaction of sequestosome-1 (p62) and LAMP1 in DARPP-32-positive medium spiny neurons in HD mice. Depletion of SUMO1 in an HD cell model also diminished the mHtt levels and enhanced autophagy flux. In addition, the SUMOylation inhibitor ginkgolic acid strongly enhanced autophagy and diminished mHTT levels in human HD fibroblasts. These results indicate that SUMO is a critical therapeutic target in HD and that blocking SUMO may ameliorate HD pathogenesis by regulating autophagy activities.

Global Rhes knockout in the Q175 Huntington's disease mouse model.

Huntington's disease (HD) results from an expansion mutation in the polyglutamine tract in huntingtin. Although huntingtin is ubiquitously expressed in the body, the striatum suffers the most severe pathology. Rhes is a Ras-related small GTP-binding protein highly expressed in the striatum that has been reported to modulate mTOR and sumoylation of mutant huntingtin to alter HD mouse model pathogenesis. Reports have varied on whether Rhes reduction is desirable for HD. Here we characterize multiple behavioral and molecular endpoints in the Q175 HD mouse model with genetic Rhes knockout (KO). Genetic RhesKO in the Q175 female mouse resulted in both subtle attenuation of Q175 phenotypic features, and detrimental effects on other kinematic features. The Q175 females exhibited measurable pathogenic deficits, as measured by MRI, MRS and DARPP32, however, RhesKO had no effect on these readouts. Additionally, RhesKO in Q175 mixed gender mice deficits did not affect mTOR signaling, autophagy or mutant huntingtin levels. We conclude that global RhesKO does not substantially ameliorate or exacerbate HD mouse phenotypes in Q175 mice.

Mutant Huntingtin stalls ribosomes and represses protein synthesis in a cellular model of Huntington disease.

The polyglutamine expansion of huntingtin (mHTT) causes Huntington disease (HD) and neurodegeneration, but the mechanisms remain unclear. Here, we found that mHtt promotes ribosome stalling and suppresses protein synthesis in mouse HD striatal neuronal cells. Depletion of mHtt enhances protein synthesis and increases the speed of ribosomal translocation, while mHtt directly inhibits protein synthesis in vitro. Fmrp, a known regulator of ribosome stalling, is upregulated in HD, but its depletion has no discernible effect on protein synthesis or ribosome stalling in HD cells. We found interactions of ribosomal proteins and translating ribosomes with mHtt. High-resolution global ribosome footprint profiling (Ribo-Seq) and mRNA-Seq indicates a widespread shift in ribosome occupancy toward the 5' and 3' end and unique single-codon pauses on selected mRNA targets in HD cells, compared to controls. Thus, mHtt impedes ribosomal translocation during translation elongation, a mechanistic defect that can be exploited for HD therapeutics.

The mammalian target of rapamycin (mTOR) kinase mediates haloperidol-induced cataleptic behavior.

The mammalian target of rapamycin (mTOR) is a ubiquitously expressed serine/threonine kinase protein complex (mTORC1 or mTORC2) that orchestrates diverse functions ranging from embryonic development to aging. However, its brain tissue-specific roles remain less explored. Here, we have identified that the depletion of the mTOR gene in the mice striatum completely prevented the extrapyramidal motor side effects (catalepsy) induced by the dopamine 2 receptor (D2R) antagonist haloperidol, which is the most widely used typical antipsychotic drug. Conversely, a lack of striatal mTOR in mice did not affect catalepsy triggered by the dopamine 1 receptor (D1R) antagonist SCH23390. Along with the lack of cataleptic effects, the administration of haloperidol in mTOR mutants failed to increase striatal phosphorylation levels of ribosomal protein pS6 (S235/236) as seen in control animals. To confirm the observations of the genetic approach, we used a pharmacological method and determined that the mTORC1 inhibitor rapamycin has a profound influence upon post-synaptic D2R-dependent functions. We consistently found that pretreatment with rapamycin entirely prevented (in a time-dependent manner) the haloperidol-induced catalepsy, and pS6K (T389) and pS6 (S235/236) signaling upregulation, in wild-type mice. Collectively, our data indicate that striatal mTORC1 blockade may offer therapeutic benefits with regard to the prevention of D2R-dependent extrapyramidal motor side effects of haloperidol in psychiatric illness.

Early motor deficits in the phalangeal fine movements induced by chronic AMPA infusion in the rat spinal cord assessed by a novel method: Phalangeal tension recording test.

Motor behavior alterations are a shared hallmark of neurodegenerative diseases affecting motor circuits, such as amyotrophic lateral sclerosis (ALS), Parkinson's, and Huntington's diseases. In patients and transgenic animal models of amyotrophic lateral sclerosis fine movements controlled by distal muscles are the first to be affected, but its study and knowledge remain poorly understood, mainly because most of the tests used for describing the motor alterations are focused on the function of proximal muscles and gross movements. In this study we demonstrate that alterations of phalangeal fine movements can be quantitatively evaluated using a novel procedure designed by us, phalangeal tension recording test, which showed high sensitivity to detect such alterations. The evaluation was carried out during the motor neuron (MN) degenerative process induced by the acute and chronic overactivation of AMPA receptors in the lumbar rat spinal cord, using previously described models. The new method allowed the quantification of significant alterations of the fine movements of the hindpaws phalanges when AMPA was infused in the lumbar segment controlling the distal muscles, but not when a more rostral spinal segment was infused, and these alterations were not detected by the rotarod or the stride tests. These changes occurred before the paralysis of the hindlimbs. Studying the early distal motor alterations before the total paralysis at late stages is essential for understanding the initial consequences of MN degeneration and therefore for designing new strategies for the control, treatment and prevention of MN diseases.

Cyclic GMP-AMP synthase promotes the inflammatory and autophagy responses in Huntington disease.

Huntington disease (HD) is caused by an expansion mutation of the N-terminal polyglutamine of huntingtin (mHTT). mHTT is ubiquitously present, but it induces noticeable damage to the brain's striatum, thereby affecting motor, psychiatric, and cognitive functions. The striatal damage and progression of HD are associated with the inflammatory response; however, the underlying molecular mechanisms remain unclear. Here, we report that cGMP-AMP synthase (cGAS), a DNA sensor, is a critical regulator of inflammatory and autophagy responses in HD. Ribosome profiling revealed that the cGAS mRNA has high ribosome occupancy at exon 1 and codon-specific pauses at positions 171 (CCG) and 172 (CGT) in HD striatal cells. Moreover, the protein levels and activity of cGAS (based on the phosphorylated STING and phosphorylated TBK1 levels), and the expression and ribosome occupancy of cGAS-dependent inflammatory genes (Ccl5 and Cxcl10) are increased in HD striatum. Depletion of cGAS diminishes cGAS activity and decreases the expression of inflammatory genes while suppressing the up-regulation of autophagy in HD cells. In contrast, reinstating cGAS in cGAS-depleted HD cells activates cGAS activity and promotes inflammatory and autophagy responses. Ribosome profiling also revealed that LC3A and LC3B, the two major autophagy initiators, show altered ribosome occupancy in HD cells. We also detected the presence of numerous micronuclei, which are known to induce cGAS, in the cytoplasm of neurons derived from human HD embryonic stem cells. Collectively, our results indicate that cGAS is up-regulated in HD and mediates inflammatory and autophagy responses. Thus, targeting the cGAS pathway may offer therapeutic benefits in HD.

RasGRP1 is a causal factor in the development of l-DOPA-induced dyskinesia in Parkinson's disease.

The therapeutic effects of l-3,4-dihydroxyphenylalanine (l-DOPA) in patients with Parkinson's disease (PD) severely diminishes with the onset of abnormal involuntary movement, l-DOPA-induced dyskinesia (LID). However, the molecular mechanisms that promote LID remain unclear. Here, we demonstrated that RasGRP1 [(guanine nucleotide exchange factor (GEF)] controls the development of LID. l-DOPA treatment rapidly up-regulated RasGRP1 in the striatum of mouse and macaque model of PD. The lack of RasGRP1 in mice (RasGRP1-/- ) dramatically diminished LID without interfering with the therapeutic effects of l-DOPA. Besides acting as a GEF for Ras homolog enriched in the brain (Rheb), the activator of the mammalian target of rapamycin kinase (mTOR), RasGRP1 promotes l-DOPA-induced extracellular signal-regulated kinase (ERK) and the mTOR signaling in the striatum. High-resolution tandem mass spectrometry analysis revealed multiple RasGRP1 downstream targets linked to LID vulnerability. Collectively, the study demonstrated that RasGRP1 is a critical striatal regulator of LID.

Rhes, a striatal-enriched protein, promotes mitophagy via Nix.

Elimination of dysfunctional mitochondria via mitophagy is essential for cell survival and neuronal functions. But, how impaired mitophagy participates in tissue-specific vulnerability in the brain remains unclear. Here, we find that striatal-enriched protein, Rhes, is a critical regulator of mitophagy and striatal vulnerability in brain. In vivo interactome and density fractionation reveal that Rhes coimmunoprecipitates and cosediments with mitochondrial and lysosomal proteins. Live-cell imaging of cultured striatal neuronal cell line shows Rhes surrounds globular mitochondria, recruits lysosomes, and ultimately degrades mitochondria. In the presence of 3-nitropropionic acid (3-NP), an inhibitor of succinate dehydrogenase, Rhes disrupts mitochondrial membrane potential (ΔΨ m ) and promotes excessive mitophagy and cell death. Ultrastructural analysis reveals that systemic injection of 3-NP in mice promotes globular mitochondria, accumulation of mitophagosomes, and striatal lesion only in the wild-type (WT), but not in the Rhes knockout (KO), striatum, suggesting that Rhes is critical for mitophagy and neuronal death in vivo. Mechanistically, Rhes requires Nix (BNIP3L), a known receptor of mitophagy, to disrupt ΔΨ m and promote mitophagy and cell death. Rhes interacts with Nix via SUMO E3-ligase domain, and Nix depletion totally abrogates Rhes-mediated mitophagy and cell death in the cultured striatal neuronal cell line. Finally, we find that Rhes, which travels from cell to cell via tunneling nanotube (TNT)-like cellular protrusions, interacts with dysfunctional mitochondria in the neighboring cell in a Nix-dependent manner. Collectively, Rhes is a major regulator of mitophagy via Nix, which may determine striatal vulnerability in the brain.

Excitatory and Inhibitory Neuronal Circuits in the Spinal Cord and Their Role in the Control of Motor Neuron Function and Degeneration.

The complex neuronal networks of the spinal cord coordinate a wide variety of motor functions, including walking, running, and voluntary and involuntary movements. This is accomplished by different groups of neurons, called center pattern generators, which control left-right alternation and flexor-extensor patterns. These spinal circuits, located in the ventral horns, are formed by several neuronal types, and the specific function of most of them has been identified by means of studies in vivo and in the isolated spinal cord of mice harboring genetically induced ablation of specific neuronal populations. These studies have shown that the coordinated activity of several interneuron types, mainly GABAergic and glycinergic inhibitory neurons, have a crucial role in the modulation of motor neurons activity that finally excites the corresponding muscles. A pharmacological experimental approach by administering in the spinal cord agonists and antagonists of glutamate, GABA, glycine, and acetylcholine receptors to alter their synaptic action has also produced important results, linking the deficits in the synaptic function with the resulting motor alterations. These results have also increased the knowledge of the mechanisms of motor neuron degeneration, which is characteristic of diseases such as amyotrophic lateral sclerosis, and therefore open the possibility of designing new strategies for the prevention and treatment of these diseases.

Chronic GABAergic blockade in the spinal cord in vivo induces motor alterations and neurodegeneration.

Inhibitory GABAergic and glycinergic neurotransmission in the spinal cord play a central role in the regulation of neuronal excitability, by maintaining a balance with the glutamate-mediated excitatory transmission. Glutamatergic agonists infusion in the spinal cord induce motor neuron death by excitotoxicity, leading to motor deficits and paralysis, but little is known on the effect of the blockade of inhibitory transmission. In this work we studied the effects of GABAergic and glycinergic blockade, by means of microdialysis perfusion (acute administration) and osmotic minipumps infusion (chronic administration) of GABA and glycine receptors antagonists directly in the lumbar spinal cord. We show that acute glycinergic blockade with strychnine or GABAergic blockade with bicuculline had no significant effects on motor activity and on motor neuron survival. However, chronic bicuculline infusion, but not strychnine, induced ipsilateral gait alterations, phalange flaccidity and significant motor neuron loss, and these effects were prevented by AMPA receptor blockade with CNQX but not by NMDA receptor blockade with MK801. In addition, we demonstrate that the chronic infusion of bicuculline enhanced the excitotoxic effect of AMPA, causing faster bilateral paralysis and increasing motor neuron loss. These findings indicate a relevant role of GABAergic inhibitory circuits in the regulation of motor neuron excitability and suggest that their alterations may be involved in the neurodegeneration processes characteristic of motor neuron diseases such as amyotrophic lateral sclerosis.

Neuropathological characterization of spinal motor neuron degeneration processes induced by acute and chronic excitotoxic stimulus in vivo.

Motor neuron (MN) diseases are characterized by progressive cell degeneration, and excitotoxicity has been postulated as a causal factor. Using two experimental procedures for inducing excitotoxic spinal MN degeneration in vivo, by acute and chronic overactivation of α-amino-3-hydroxy-5-methyl-4-isoxazoleacetic acid (AMPA) receptors, we characterized the time course of the neuropathological changes. Electron transmission microscopy showed that acute AMPA perfusion by microdialysis caused MN swelling 1.5h after surgery and lysis with membrane rupture as early as 3h; no cleaved caspase 3 was detected by immunochemistry. Chronic AMPA infusion by osmotic minipumps induced a slow degeneration process along 5days, characterized by progressive changes: endoplasmic reticulum swelling, vacuolization of cytoplasm, vacuole fusion and cell membrane rupture. Quantification of these ultrastructural alterations showed that the increase of vacuolated area was at the expense of the nuclear area. Caspase 3 cleavage was observed since the first day of AMPA infusion. We conclude that acute AMPA-induced excitotoxicity induces MN loss by necrosis, while the progress of degeneration induced by chronic infusion is slow, starting with an early apoptotic process followed by necrosis. In both the acute and chronic procedures a correlation could be established between the loss of MN by necrosis, but not by caspase 3-linked apoptosis, and severe motor deficits and hindlimb paralysis. Our findings are relevant for understanding the mechanisms of neuron death in degenerative diseases and thus for the design of pharmacological therapeutic strategies.

Trophic factors as modulators of motor neuron physiology and survival: implications for ALS therapy.

Motor neuron physiology and development depend on a continuous and tightly regulated trophic support from a variety of cellular sources. Trophic factors guide the generation and positioning of motor neurons during every stage of the developmental process. As well, they are involved in axon guidance and synapse formation. Even in the adult spinal cord an uninterrupted trophic input is required to maintain neuronal functioning and protection from noxious stimuli. Among the trophic factors that have been demonstrated to participate in motor neuron physiology are vascular endothelial growth factor (VEGF), glial-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF) and insulin-like growth factor 1 (IGF-1). Upon binding to membrane receptors expressed in motor neurons or neighboring glia, these trophic factors activate intracellular signaling pathways that promote cell survival and have protective action on motor neurons, in both in vivo and in vitro models of neuronal degeneration. For these reasons these factors have been considered a promising therapeutic method for amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases, although their efficacy in human clinical trials have not yet shown the expected protection. In this minireview we summarize experimental data on the role of these trophic factors in motor neuron function and survival, as well as their mechanisms of action. We also briefly discuss the potential therapeutic use of the trophic factors and why these therapies may have not been yet successful in the clinical use.

Spinal inhibitory circuits and their role in motor neuron degeneration.

In the spinal cord neuronal activity is controlled by the balance between excitatory and inhibitory neurotransmission, mediated mainly by the neurotransmitters glutamate and GABA/glycine, respectively. Alterations of this equilibrium have been associated with spinal motor neuron hyperexcitability and degeneration, which can be induced by excitotoxicity or by decreasing inhibitory neurotransmission. Here we review the ventral horn neuronal network and the possible involvement of inhibitory circuits in the mechanisms of degeneration of motor neurons characteristic of amyotrophic lateral sclerosis (ALS). Whereas glutamate mediated excitotoxicity seems to be an important factor, recent experimental and histopathological evidence argue in favor of a decreased activity of the inhibitory circuits controlling motor neuron excitability, mainly the recurrent inhibition exerted by Renshaw cells. A decreased Renshaw cell activity may be caused by cell loss or by a reduction of its inhibitory action secondary to a decreased excitation from cholinergic interneurons. Ultimately, inhibitory failure by either mechanism might lead to motor neuron degeneration, and this suggests inhibitory circuits and Renshaw cells as pharmacologic targets for ALS treatment.