Automated in vivo drug screen in zebrafish identifies synapse-stabilising drugs with relevance to spinal muscular atrophy.

Synapses are particularly vulnerable in many neurodegenerative diseases and often the first to degenerate, for example in the motor neuron disease spinal muscular atrophy (SMA). Compounds that can counteract synaptic destabilisation are rare. Here, we describe an automated screening paradigm in zebrafish for small-molecule compounds that stabilize the neuromuscular synapse in vivo. We make use of a mutant for the axonal C-type lectin chondrolectin (chodl), one of the main genes dysregulated in SMA. In chodl-/- mutants, neuromuscular synapses that are formed at the first synaptic site by growing axons are not fully mature, causing axons to stall, thereby impeding further axon growth beyond that synaptic site. This makes axon length a convenient read-out for synapse stability. We screened 982 small-molecule compounds in chodl chodl-/- mutants and found four that strongly rescued motor axon length. Aberrant presynaptic neuromuscular synapse morphology was also corrected. The most-effective compound, the adenosine uptake inhibitor drug dipyridamole, also rescued axon growth defects in the UBA1-dependent zebrafish model of SMA. Hence, we describe an automated screening pipeline that can detect compounds with relevance to SMA. This versatile platform can be used for drug and genetic screens, with wider relevance to synapse formation and stabilisation.

Neuroprotective activity of ursodeoxycholic acid in CHMP2BIntron5 models of frontotemporal dementia.

Frontotemporal dementia (FTD) is one of the most prevalent forms of early-onset dementia. It represents part of the FTD-Amyotrophic Lateral Sclerosis (ALS) spectrum, a continuum of genetically and pathologically overlapping disorders. FTD-causing mutations in CHMP2B, a gene encoding a core component of the heteromeric ESCRT-III Complex, lead to perturbed endosomal-lysosomal and autophagic trafficking with impaired proteostasis. While CHMP2B mutations are rare, dysfunctional endosomal-lysosomal signalling is common across the FTD-ALS spectrum. Using our established Drosophila and mammalian models of CHMP2BIntron5 induced FTD we demonstrate that the FDA-approved compound Ursodeoxycholic Acid (UDCA) conveys neuroprotection, downstream of endosomal-lysosomal dysfunction in both Drosophila and primary mammalian neurons. UDCA exhibited a dose dependent rescue of neuronal structure and function in Drosophila pan-neuronally expressing CHMP2BIntron5. Rescue of CHMP2BIntron5 dependent dendritic collapse and apoptosis with UDCA in rat primary neurons was also observed. UDCA failed to ameliorate aberrant accumulation of endosomal and autophagic organelles or ubiquitinated neuronal inclusions in both models. We demonstrate the neuroprotective activity of UDCA downstream of endosomal-lysosomal and autophagic dysfunction, delineating the molecular mode of action of UDCA and highlighting its potential as a therapeutic for the treatment of FTD-ALS spectrum disorders.

Passive Transfer of Sera from ALS Patients with Identified Mutations Evokes an Increased Synaptic Vesicle Number and Elevation of Calcium Levels in Motor Axon Terminals, Similar to Sera from Sporadic Patients.

Previously, we demonstrated increased calcium levels and synaptic vesicle densities in the motor axon terminals (MATs) of sporadic amyotrophic lateral sclerosis (ALS) patients. Such alterations could be conferred to mice with an intraperitoneal injection of sera from these patients or with purified immunoglobulin G. Later, we confirmed the presence of similar alterations in the superoxide dismutase 1 G93A transgenic mouse strain model of familial ALS. These consistent observations suggested that calcium plays a central role in the pathomechanism of ALS. This may be further reinforced by completing a similar analytical study of the MATs of ALS patients with identified mutations. However, due to the low yield of muscle biopsy samples containing MATs, and the low incidence of ALS patients with the identified mutations, these examinations are not technically feasible. Alternatively, a passive transfer of sera from ALS patients with known mutations was used, and the MATs of the inoculated mice were tested for alterations in their calcium homeostasis and synaptic activity. Patients with 11 different ALS-related mutations participated in the study. Intraperitoneal injection of sera from these patients on two consecutive days resulted in elevated intracellular calcium levels and increased vesicle densities in the MATs of mice, which is comparable to the effect of the passive transfer from sporadic patients. Our results support the idea that the pathomechanism underlying the identical manifestation of the disease with or without identified mutations is based on a common final pathway, in which increasing calcium levels play a central role.

Presynaptic Caytaxin prevents apoptosis via deactivating DAPK1 in the acute phase of cerebral ischemic stroke.

Death-associated protein kinase 1 (DAPK1) is a key protein that mediates neuronal death in ischemic stroke. Although the substrates of DAPK1 and molecular signal in stroke have been gradually discovered, the modulation of DAPK1 itself is still unclear. Here we first reveal that Caytaxin, a brain-specific member of BCL2/adenovirus E1B -interacting protein (BNIP-2), increases and interacts with DAPK1 as early as 2 h after middle cerebral artery occlusion (MCAO) in the penumbra area of mouse brain. Furthermore, Caytaxin binds to DAPK1 at the presynaptic site and inhibits DAPK1 catalytic activity. Silencing Caytaxin by Caytaxin shRNA (Sh-Caytaxin) enhances DAPK1 activity, deteriorates neuronal apoptosis and brain injuries both in vivo and in vitro. Thus, elevating presynaptic Caytaxin could prevent neuronal apoptosis by inhibiting DAPK1 activation in the acute stage of ischemic stroke. Caytaxin may physiologically protect neuronal cells and represent a potential prevention and therapeutic target in the early phase of cerebral ischemic stroke.

NP03, a Microdose Lithium Formulation, Blunts Early Amyloid Post-Plaque Neuropathology in McGill-R-Thy1-APP Alzheimer-Like Transgenic Rats.

Epidemiological, preclinical, and clinical studies have suggested a role for microdose lithium in reducing Alzheimer's disease (AD) risk by modulating key mechanisms associated with AD pathology. The novel microdose lithium formulation, NP03, has disease-modifying effects in the McGill-R-Thy1-APP transgenic rat model of AD-like amyloidosis at pre-plaque stages, before frank amyloid-ß (Aß) plaque deposition, during which Aß is primarily intraneuronal. Here, we are interested in determining whether the positive effects of microdose lithium extend into early Aß post-plaque stages. We administered NP03 (40µg Li/kg; 1 ml/kg body weight) to McGill-R-Thy1-APP transgenic rats for 12 weeks spanning the transition phase from plaque-free to plaque-bearing. The effect of NP03 on remote working memory was assessed using the novel object recognition task. Levels of human Aß38, Aß40, and Aß42 as well as levels of pro-inflammatory mediators were measured in brain-extracts and plasma using electrochemiluminescent assays. Mature Aß plaques were visualized with a thioflavin-S staining. Vesicular acetylcholine transporter (VAChT) bouton density and levels of chemokine (C-X-C motif) ligand 1 (CXCL1), interleukin-6 (IL-6), and 4-hydroxynonenal (4-HNE) were probed using quantitative immunohistochemistry. During the early Aß post-plaque stage, we find that NP03 rescues functional deficits in object recognition, reduces loss of cholinergic boutons in the hippocampus, reduces levels of soluble and insoluble cortical Aß42 and reduces hippocampal Aß plaque number. In addition, NP03 reduces markers of neuroinflammation and cellular oxidative stress. Together these results indicate that microdose lithium NP03 is effective at later stages of amyloid pathology, after appearance of Aß plaques.

Environmental training is beneficial to clinical symptoms and cortical presynaptic defects in mice suffering from experimental autoimmune encephalomyelitis.

The effect of "prophylactic" environmental stimulation on clinical symptoms and presynaptic defects in mice suffering from the experimental autoimmune encephalomyelitis (EAE) at the acute stage of disease (21 ±â€¯1 days post immunization, d.p.i.) was investigated. In EAE mice raised in an enriched environment (EE), the clinical score was reduced when compared to EAE mice raised in standard environment (SE).Concomitantly, gain of weight and increased spontaneous motor activity and curiosity were observed, suggesting increased well-being in mice. Impaired glutamate exocytosis and cyclic adenosine monophosphate (cAMP) production in cortical terminals of SE-EAE mice were evident at 21 ±â€¯1 d.p.i.. Differently, the 12 mM KCl-evoked glutamate exocytosis from cortical synaptosomes of EE-EAE mice was comparable to that observed in SE and EE-control mice, but significantly higher than that in SE-EAE mice. Similarly, the 12 mM KCl-evoked cAMP production in EE-EAE mice cortical synaptosomes recovered to the level observed in SE and EE-control mice. MUNC-18 and SNAP25 contents, but not Syntaxin-1a and Synaptotagmin 1 levels, were increased in cortical synaptosomes from EE-EAE mice when compared to SE-EAE mice. Circulating IL-1ß was increased in the spinal cord, but not in the cortex, of SE-EAE mice, and it did not recover in EE-EAE mice. Inflammatory infiltrates were reduced in the cortex but not in the spinal cord of EE-EAE mice. Demyelination was observed in the spinal cord; EE significantly diminished it. We conclude that "prophylactic" EE is beneficial to synaptic derangements and preserves glutamate transmission in the cortex of EAE mice. This article is part of the Special Issue entitled "Neurobiology of Environmental Enrichment".

Intracellular Ca2+ stores control in vivo neuronal hyperactivity in a mouse model of Alzheimer's disease.

Neuronal hyperactivity is the emerging functional hallmark of Alzheimer's disease (AD) in both humans and different mouse models, mediating an impairment of memory and cognition. The mechanisms underlying neuronal hyperactivity remain, however, elusive. In vivo Ca2+ imaging of somatic, dendritic, and axonal activity patterns of cortical neurons revealed that both healthy aging and AD-related mutations augment neuronal hyperactivity. The AD-related enhancement occurred even without amyloid deposition and neuroinflammation, mainly due to presenilin-mediated dysfunction of intracellular Ca2+ stores in presynaptic boutons, likely causing more frequent activation of synaptic NMDA receptors. In mutant but not wild-type mice, store emptying reduced both the frequency and amplitude of presynaptic Ca2+ transients and, most importantly, normalized neuronal network activity. Postsynaptically, the store dysfunction was minor and largely restricted to hyperactive cells. These findings identify presynaptic Ca2+ stores as a key element controlling AD-related neuronal hyperactivity and as a target for disease-modifying treatments.

Erythropoietin prevents the effect of chronic restraint stress on the number of hippocampal CA3c dendritic terminals-relation to expression of genes involved in synaptic plasticity, angiogenesis, inflammation, and oxidative stress in male rats.

Stress-induced allostatic load affects a variety of biological processes including synaptic plasticity, angiogenesis, oxidative stress, and inflammation in the brain, especially in the hippocampus. Erythropoietin (EPO) is a pleiotropic cytokine that has shown promising neuroprotective effects. Recombinant human EPO is currently highlighted as a new candidate treatment for cognitive impairment in neuropsychiatric disorders. Because EPO enhances synaptic plasticity, attenuates oxidative stress, and inhibits generation of proinflammatory cytokines, EPO may be able to modulate the effects of stress-induced allostatic load at the molecular level. The aim of this study was therefore to investigate how EPO and repeated restraint stress, separately and combined, influence (i) behavior in the novelty-suppressed feeding test of depression/anxiety-related behavior; (ii) mRNA levels of genes encoding proteins involved in synaptic plasticity, angiogenesis, oxidative stress, and inflammation; and (iii) remodeling of the dendritic structure of the CA3c area of the hippocampus in male rats. As expected, chronic restraint stress lowered the number of CA3c apical dendritic terminals, and EPO treatment reversed this effect. Interestingly, these effects seemed to be mechanistically distinct, as stress and EPO had differential effects on gene expression. While chronic restraint stress lowered the expression of spinophilin, tumor necrosis factor α, and heat shock protein 72, EPO increased expression of hypoxia-inducible factor-2α and lowered the expression of vascular endothelial growth factor in hippocampus. These findings indicate that the effects of treatment with EPO follow different molecular pathways and do not directly counteract the effects of stress in the hippocampus.

The Role of Cysteine String Protein α Phosphorylation at Serine 10 and 34 by Protein Kinase Cγ for Presynaptic Maintenance.

Protein kinase Cγ (PKCγ) knock-out (KO) animals exhibit symptoms of Parkinson's disease (PD), including dopaminergic neuronal loss in the substantia nigra. However, the PKCγ substrates responsible for the survival of dopaminergic neurons in vivo have not yet been elucidated. Previously, we found 10 potent substrates in the striatum of PKCγ-KO mice. Here, we focused on cysteine string protein α (CSPα), a protein from the heat shock protein (HSP) 40 cochaperone families localized on synaptic vesicles. We found that in cultured cells, PKCγ phosphorylates CSPα at serine (Ser) 10 and Ser34. Additionally, apoptosis was found to have been enhanced by the overexpression of a phosphorylation-null mutant of CSPα, CSPα(S10A/S34A). Compared with wild-type (WT) CSPα, the CSPα(S10A/S34A) mutant had a weaker interaction with HSP70. However, in sharp contrast, a phosphomimetic CSPα(S10D/S34D) mutant, compared with WT CSPα, had a stronger interaction with HSP70. In addition, total levels of synaptosomal-associated protein (SNAP) 25, a main downstream target of the HSC70/HSP70 chaperone complex, were found to have decreased by the CSPα(S10A/S34A) mutant through increased ubiquitination of SNAP25 in PC12 cells. In the striatum of 2-year-old male PKCγ-KO mice, decreased phosphorylation levels of CSPα and decreased SNAP25 protein levels were observed. These findings indicate the phosphorylation of CSPα by PKCγ may protect the presynaptic terminal from neurodegeneration. The PKCγ-CSPα-HSC70/HSP70-SNAP25 axis, because of its role in protecting the presynaptic terminal, may provide a new therapeutic target for the treatment of PD.SIGNIFICANCE STATEMENT Cysteine string protein α (CSPα) is a protein belonging to the heat shock protein (HSP) 40 cochaperone families localized on synaptic vesicles, which maintain the presynaptic terminal. However, the function of CSPα phosphorylation by protein kinase C (PKC) for neuronal cell survival remains unclear. The experiments presented here demonstrate that PKCγ phosphorylates CSPα at serine (Ser) 10 and Ser34. CSPα phosphorylation at Ser10 and Ser34 by PKCγ protects the presynaptic terminal by promoting HSP70 chaperone activity. This report suggests that CSPα phosphorylation, because of its role in modulating HSP70 chaperone activity, may be a target for the treatment of neurodegeneration.

Central nervous system-penetrating antiretrovirals impair energetic reserve in striatal nerve terminals.

The use of antiretroviral (ARV) drugs with central nervous system (CNS) penetration effectiveness (CPE) may be useful in the treatment of HIV-associated neurocognitive disorder (HAND) as well as targeting a CNS reservoir in strategies to achieve a functional cure for HIV. However, increased cognitive deficits are linked to at least one of these drugs (efavirenz). As mitochondrial dysfunction has been found with a number of ARVs, and as such can affect neuronal function, the objective of this study was to assess the effects of ARV with high CPE for toxicological profiles on presynaptic nerve terminal energy metabolism. This subcellular region is especially vulnerable in that a constant supply of ATP is required for the proper maintenance of neurotransmitter release and uptake supporting proper neuronal function. We evaluated the effects of acute treatment with ten different high CPE ARVs from five different drug classes on rat cortical and striatal nerve terminal bioenergetic function. While cortical nerve terminal bioenergetics were not altered, striatal nerve terminals exposed to efavirenz, nevirapine, abacavir, emtricitabine, zidovudine, darunavir, lopinavir, raltegravir, or maraviroc (but not indinavir) exhibit reduced mitochondrial spare respiratory capacity (SRC). Further examination of efavirenz and maraviroc revealed a concentration-dependent impairment of striatal nerve terminal maximal mitochondrial respiration and SRC as well as a reduction of intraterminal ATP levels. Depletion of ATP at the synapse may underlie its dysfunction and contribute to neuronal dysfunction in treated HIV infection.

Early synaptic dysfunction induced by α-synuclein in a rat model of Parkinson's disease.

Evidence suggests that synapses are affected first in Parkinson's disease (PD). Here, we tested the claim that pathological accumulation of α-synuclein, and subsequent synaptic disruption, occur in absence of dopaminergic neuron loss in PD. We determined early synaptic changes in rats that overexpress human α-synuclein by local injection of viral-vectors in midbrain. We aimed to achieve α-synuclein levels sufficient to induce terminal pathology without significant loss of nigral neurons. We tested synaptic disruption in vivo by analyzing motor defects and binding of a positron emission tomography (PET) radioligand to the vesicular monoamine transporter 2, (VMAT2), [11C]dihydrotetrabenazine (DTBZ). Animals overexpressing α-synuclein had progressive motor impairment and, 12 weeks post-surgery, showed asymmetric in vivo striatal DTBZ binding. The PET images matched ligand binding in post-mortem tissue, and histological markers of dopaminergic integrity. Histology confirmed the absence of nigral cell death with concomitant significant loss of striatal terminals. Progressive aggregation of proteinase-K resistant and Ser129-phosphorylated α-synuclein was observed in dopaminergic terminals, in dystrophic swellings that resembled axonal spheroids and contained mitochondria and vesicular proteins. In conclusion, pathological α-synuclein in nigro-striatal axonal terminals leads to early axonal pathology, synaptic disruption, dysfunction of dopaminergic neurotransmission, motor impairment, and measurable change of VMAT2 in the absence of cell loss.

Bortezomib induces neuropathic pain through protein kinase C-mediated activation of presynaptic NMDA receptors in the spinal cord.

Chemotherapeutic drugs, including bortezomib, often cause painful peripheral neuropathy, which is a severe dose-limiting adverse effect experienced by many cancer patients. The glutamate N-methyl-d-aspartate receptors (NMDARs) at the spinal cord level are critically involved in the synaptic plasticity associated with neuropathic pain. In this study, we determined whether treatment with bortezomib, a proteasome inhibitor, affects the NMDAR activity of spinal dorsal horn neurons. Systemic treatment with bortezomib in rats did not significantly affect postsynaptic NMDAR currents elicited by puff application of NMDA directly to dorsal horn neurons. Bortezomib treatment markedly increased the baseline frequency of miniature excitatory postsynaptic currents (EPSCs), which was completely normalized by the NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5). AP5 also reduced the amplitude of monosynaptic EPSCs evoked by dorsal root stimulation in bortezomib-treated, but not vehicle-treated, rats. Furthermore, inhibition of protein kinase C (PKC) with chelerythrine fully reversed the increased frequency of miniature EPSCs and the amplitude of evoked EPSCs in bortezomib-treated rats. Intrathecal injection of AP5 and chelerythrine both profoundly attenuated mechanical allodynia and hyperalgesia induced by systemic treatment with bortezomib. In addition, treatment with bortezomib induced striking membrane translocation of PKC-ßII, PKC-δ, and PKC-ε in the dorsal root ganglion. Our findings indicate that bortezomib treatment potentiates nociceptive input from primary afferent nerves via PKC-mediated tonic activation of presynaptic NMDARs. Targeting presynaptic NMDARs and PKC at the spinal cord level may be an effective strategy for treating chemotherapy-induced neuropathic pain.

KCC3 loss-of-function contributes to Andermann syndrome by inducing activity-dependent neuromuscular junction defects.

Loss-of-function mutations in the potassium-chloride cotransporter KCC3 lead to Andermann syndrome, a severe sensorimotor neuropathy characterized by areflexia, amyotrophy and locomotor abnormalities. The molecular events responsible for axonal loss remain poorly understood. Here, we establish that global or neuron-specific KCC3 loss-of-function in mice leads to early neuromuscular junction (NMJ) abnormalities and muscular atrophy that are consistent with the pre-synaptic neurotransmission defects observed in patients. KCC3 depletion does not modify chloride handling, but promotes an abnormal electrical activity among primary motoneurons and mislocalization of Na+/K+-ATPase α1 in spinal cord motoneurons. Moreover, the activity-targeting drug carbamazepine restores Na+/K+-ATPase α1 localization and reduces NMJ denervation in Slc12a6-/- mice. We here propose that abnormal motoneuron electrical activity contributes to the peripheral neuropathy observed in Andermann syndrome.

Free radical production and antioxidant status in brain cortex non-synaptic mitochondria and synaptosomes at alcohol hangover onset.

Alcohol hangover (AH) is the pathophysiological state after a binge-like drinking. We have previously demonstrated that AH induced bioenergetics impairments in a total fresh mitochondrial fraction in brain cortex and cerebellum. The aim of this work was to determine free radical production and antioxidant systems in non-synaptic mitochondria and synaptosomes in control and hangover animals. Superoxide production was not modified in non-synaptic mitochondria while a 17.5% increase was observed in synaptosomes. A similar response was observed for cardiolipin content as no changes were evidenced in non-synaptic mitochondria while a 55% decrease in cardiolipin content was found in synaptosomes. Hydrogen peroxide production was 3-fold increased in non-synaptic mitochondria and 4-fold increased in synaptosomes. In the presence of deprenyl, synaptosomal H2O2 production was 67% decreased in the AH condition. Hydrogen peroxide generation was not affected by deprenyl addition in non-synaptic mitochondria from AH mice. MAO activity was 57% increased in non-synaptic mitochondria and 3-fold increased in synaptosomes. Catalase activity was 40% and 50% decreased in non-synaptic mitochondria and synaptosomes, respectively. Superoxide dismutase was 60% decreased in non-synaptic mitochondria and 80% increased in synaptosomal fractions. On the other hand, GSH (glutathione) content was 43% and 17% decreased in synaptosomes and cytosol. GSH-related enzymes were mostly affected in synaptosomes fractions by AH condition. Acetylcholinesterase activity in synaptosomes was 11% increased due to AH. The present work reveals that AH provokes an imbalance in the cellular redox homeostasis mainly affecting mitochondria present in synaptic terminals.

Lysosomal dysfunction disrupts presynaptic maintenance and restoration of presynaptic function prevents neurodegeneration in lysosomal storage diseases.

Lysosomal storage disorders (LSDs) are inherited diseases characterized by lysosomal dysfunction and often showing a neurodegenerative course. There is no cure to treat the central nervous system in LSDs. Moreover, the mechanisms driving neuronal degeneration in these pathological conditions remain largely unknown. By studying mouse models of LSDs, we found that neurodegeneration develops progressively with profound alterations in presynaptic structure and function. In these models, impaired lysosomal activity causes massive perikaryal accumulation of insoluble α-synuclein and increased proteasomal degradation of cysteine string protein α (CSPα). As a result, the availability of both α-synuclein and CSPα at nerve terminals strongly decreases, thus inhibiting soluble NSF attachment receptor (SNARE) complex assembly and synaptic vesicle recycling. Aberrant presynaptic SNARE phenotype is recapitulated in mice with genetic ablation of one allele of both CSPα and α-synuclein. The overexpression of CSPα in the brain of a mouse model of mucopolysaccharidosis type IIIA, a severe form of LSD, efficiently re-established SNARE complex assembly, thereby ameliorating presynaptic function, attenuating neurodegenerative signs, and prolonging survival. Our data show that neurodegenerative processes associated with lysosomal dysfunction may be presynaptically initiated by a concomitant reduction in α-synuclein and CSPα levels at nerve terminals. They also demonstrate that neurodegeneration in LSDs can be slowed down by re-establishing presynaptic functions, thus identifying synapse maintenance as a novel potentially druggable target for brain treatment in LSDs.

Impaired Presynaptic High-Affinity Choline Transporter Causes a Congenital Myasthenic Syndrome with Episodic Apnea.

The neuromuscular junction (NMJ) is one of the best-studied cholinergic synapses. Inherited defects of peripheral neurotransmission result in congenital myasthenic syndromes (CMSs), a clinically and genetically heterogeneous group of rare diseases with fluctuating fatigable muscle weakness as the clinical hallmark. Whole-exome sequencing and Sanger sequencing in six unrelated families identified compound heterozygous and homozygous mutations in SLC5A7 encoding the presynaptic sodium-dependent high-affinity choline transporter 1 (CHT), which is known to be mutated in one dominant form of distal motor neuronopathy (DHMN7A). We identified 11 recessive mutations in SLC5A7 that were associated with a spectrum of severe muscle weakness ranging from a lethal antenatal form of arthrogryposis and severe hypotonia to a neonatal form of CMS with episodic apnea and a favorable prognosis when well managed at the clinical level. As expected given the critical role of CHT for multisystemic cholinergic neurotransmission, autonomic dysfunctions were reported in the antenatal form and cognitive impairment was noticed in half of the persons with the neonatal form. The missense mutations induced a near complete loss of function of CHT activity in cell models. At the human NMJ, a delay in synaptic maturation and an altered maintenance were observed in the antenatal and neonatal forms, respectively. Increased synaptic expression of butyrylcholinesterase was also observed, exposing the dysfunction of cholinergic metabolism when CHT is deficient in vivo. This work broadens the clinical spectrum of human diseases resulting from reduced CHT activity and highlights the complexity of cholinergic metabolism at the synapse.

Presynaptic Disorders: Lambert-Eaton Myasthenic Syndrome and Botulism.

Lambert-Eaton myasthenic syndrome (LEMS) and botulism are acquired presynaptic nerve terminal disorders of the neuromuscular junction. Lambert-Eaton myasthenic syndrome is an idiopathic or paraneoplastic autoimmune syndrome in which autoantibodies of the P/Q-type voltage-gated calcium channel play a role in decreasing the release of acetylcholine, resulting in clinical symptoms of skeletal muscle weakness, diminished reflexes, and autonomic symptoms. Paraneoplastic LEMS is most often associated with small cell lung cancer. Diagnosis is confirmed by positive serologic testing and electrophysiological studies, which display characteristic features of low compound muscle action potentials, a decrement at 3Hz repetitive nerve stimulation, and facilitation with exercise or high-frequency repetitive stimulation. Treatment involves cancer monitoring and treatment, 3,4-diaminopyridine, immunosuppressive medications, and acetylcholinesterase inhibitors. Botulism is another presynaptic disorder of neuromuscular transmission. Clinical features classically involve cranial and bulbar palsies followed by descending weakness of the limbs, respiratory failure, and autonomic dysfunction. Electrodiagnostic testing is important in the evaluation and diagnosis. Treatment is supportive, and administration of antitoxin is beneficial in selected cases.

Synaptic mutant huntingtin inhibits synapsin-1 phosphorylation and causes neurological symptoms.

Many genetic mouse models of Huntington's disease (HD) have established that mutant huntingtin (htt) accumulates in various subcellular regions to affect a variety of cellular functions, but whether and how synaptic mutant htt directly mediates HD neuropathology remains to be determined. We generated transgenic mice that selectively express mutant htt in the presynaptic terminals. Although it was not overexpressed, synaptic mutant htt caused age-dependent neurological symptoms and early death in mice as well as defects in synaptic neurotransmitter release. Mass spectrometry analysis of synaptic fractions and immunoprecipitation of synapsin-1 from HD CAG150 knockin mouse brains revealed that mutant htt binds to synapsin-1, a protein whose phosphorylation is critical for neurotransmitter release. We found that polyglutamine-expanded exon1 htt binds to the C-terminal region of synapsin-1 to reduce synapsin-1 phosphorylation. Our findings point to a critical role for synaptic htt in the neurological symptoms of HD, providing a new therapeutic target.

Peripheral elevation of TNF-α leads to early synaptic abnormalities in the mouse somatosensory cortex in experimental autoimmune encephalomyelitis.

Sensory abnormalities such as numbness and paresthesias are often the earliest symptoms in neuroinflammatory diseases including multiple sclerosis. The increased production of various cytokines occurs in the early stages of neuroinflammation and could have detrimental effects on the central nervous system, thereby contributing to sensory and cognitive deficits. However, it remains unknown whether and when elevation of cytokines causes changes in brain structure and function under inflammatory conditions. To address this question, we used a mouse model for experimental autoimmune encephalomyelitis (EAE) to examine the effect of inflammation and cytokine elevation on synaptic connections in the primary somatosensory cortex. Using in vivo two-photon microscopy, we found that the elimination and formation rates of dendritic spines and axonal boutons increased within 7 d of EAE induction--several days before the onset of paralysis--and continued to rise during the course of the disease. This synaptic instability occurred before T-cell infiltration and microglial activation in the central nervous system and was in conjunction with peripheral, but not central, production of TNF-α. Peripheral administration of a soluble TNF inhibitor prevented abnormal turnover of dendritic spines and axonal boutons in presymptomatic EAE mice. These findings indicate that peripheral production of TNF-α is a key mediator of synaptic instability in the primary somatosensory cortex and may contribute to sensory and cognitive deficits seen in autoimmune diseases.

Mitochondrial involvement in neurodegenerative diseases.

The classical bioenergetical view of the involvement of mitochondria in neurogeneration is based on the fact that mitochondria are the central players of ATP synthesis in neurons and their failure leads to neuronal dysfunction and eventually to cell death. Mutations in at least 39 genes in inherited neurodegenerative disorders seem to alter directly or indirectly mitochondrial function. Most of these mutations do not directly affect oxidative phosphorylation, but act through disturbed mitochondrial dynamics and quality control. This, however, does not invalidate the bioenergetic hypothesis. Neurodegeneration is not necessarily associated with a gross failure of ATP production, but might rather be a consequence of local insufficiencies of ATP supply in critical compartments of neurons, like the presynaptic terminal. We hypothesize that slow disease progression, at least in a subgroup of neurodegenerative diseases, can be explained by the parallel action of subcellular ATP insufficiency and clonal expansion of somatic mitochondrial DNA mutations, and particularly deletions.