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OBJECTIVE Stereotactic radiofrequency pallidotomy has demonstrated improvement in motor fluctuations in patients with Parkinson’s disease (PD), particularly levodopa (L-dopa)–induced dyskinesias. The authors aimed to determine whether or not unilateral pallidotomy with MR-guided focused ultrasound (MRgFUS) could safely improve Unified Dyskinesia Rating Scale (UDysRS; the primary outcome measure) scores over baseline scores in patients with PD. METHODS Twenty patients with PD and L-dopa responsiveness, asymmetrical motor signs, and motor fluctuations, including dyskinesias, participated in a 1-year multicenter open-label trial of unilateral MRgFUS ablation of the globus pallidus internus. RESULTS The sonication procedure was successfully completed in all 20 enrolled patients. MRgFUS-related adverse neurological events were generally mild and transient, including visual field deficit (n = 1), dysarthria (n = 4, 2 mild and 2 moderate), cognitive disturbance (n = 1), fine motor deficit (n = 2), and facial weakness (n = 1). Although 3 adverse events (AEs) were rated as severe (transient sonication-related pain in 2, nausea/vomiting in 1), no AE fulfilled US FDA criteria for a Serious Adverse Effect. Total UDysRS, the primary outcome measure, improved 59% after treatment (baseline mean score 36.1, 95% CI 4.88; at 3 months 14.2, 95% CI 5.72, p < 0.0001), which was sustained throughout the study (at 12 months 20.5, 95% CI 7.39, 43% improvement, p < 0.0001). The severity of motor signs on the treated side (Movement Disorder Society version of the United Parkinson’s Disease Rating Scale [MDS-UPDRS] part III) in the “off” medication state also significantly improved (baseline mean score 20.0, 95% CI 2.4; at 3 months 10.6, 95% CI 1.86, 44.5% improvement, p < 0.0001; at 12 months 10.4, 95% CI 2.11, 45.2% improvement, p > 0.0001). The vast majority of patients showed a clinically meaningful level of improvement on the impairment component of the UDysRS or the motor component of the UPDRS, while 1 patient showed clinically meaningful worsening on the UPDRS at month 3. CONCLUSIONS This study supports the feasibility and preliminary efficacy of MRgFUS pallidotomy in the treatment of patients with PD and motor fluctuations, including dyskinesias. These preliminary data support continued investigation, and a placebo-controlled, blinded trial is in progress. Clinical trial registration no.: NCT02263885 (clinicaltrials.gov)

The blood brain barrier (BBB) is an obstacle for the delivery of potential molecular therapies for neurodegenerative diseases such as Parkinson's disease (PD), Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS). Although there has been a proliferation of potential disease modifying therapies for these progressive conditions, strategies to deliver these large agents remain limited. High intensity MRI guided focused ultrasound has already been FDA approved to lesion brain targets to treat movement disorders, while lower intensity pulsed ultrasound coupled with microbubbles commonly used as contrast agents can create transient safe opening of the BBB. Pre-clinical studies have successfully delivered growth factors, antibodies, genes, viral vectors, and nanoparticles in rodent models of AD and PD. Recent small clinical trials support the safety and feasibility of this strategy in these vulnerable patients. Further study is needed to establish safety as MRI guided BBB opening is used to enhance the delivery of newly developed molecular therapies.

Background Magnetic resonance imaging-guided focused ultrasound thalamotomy is approved by the U.S. Food and Drug Administration for treatment of essential tremor. Although this incisionless technology creates an ablative lesion, it potentially avoids serious complications of open stereotactic surgery. Objective To determine the safety profile of magnetic resonance imaging-guided focused ultrasound unilateral thalamotomy for essential tremor, including frequency, and severity of adverse events, including serious adverse events. Methods Analysis of safety data for magnetic resonance imaging-guided focused ultrasound thalamotomy (186 patients, five studies). Results Procedure-related serious adverse events were very infrequent (1.6%), without intracerebral hemorrhages or infections. Adverse events were usually transient and were commonly rated as mild (79%) and rarely severe (1%). As previously reported, abnormalities in sensation and balance were the commonest thalamotomy-related adverse events. Conclusion The overall safety profile of magnetic resonance imaging-guided focused ultrasound thalamotomy supports its role as a new option for patients with medically refractory essential tremor. © 2018 International Parkinson and Movement Disorder Society.

OBJECTIVE Magnetic resonance guided focused ultrasound (MRgFUS) has recently been investigated as a new treatment modality for essential tremor (ET), but the durability of the procedure has not yet been evaluated. This study reports results at a 2- year follow-up after MRgFUS thalamotomy for ET. METHODS A total of 76 patients with moderate-to-severe ET, who had not responded to at least two trials of medical therapy, were enrolled in the original randomized study of unilateral thalamotomy and evaluated using the clinical rating scale for tremor. Sixty-seven of the patients continued in the open-label extension phase of the study with monitoring for 2 years. Nine patients were excluded by 2 years, for example, because of alternative therapy such as deep brain stimulation (n = 3) or inadequate thermal lesioning (n = 1). However, all patients in each follow-up period were analyzed. RESULTS Mean hand tremor score at baseline (19.8 ± 4.9; 76 patients) improved by 55% at 6 months (8.6 ± 4.5; 75 patients). The improvement in tremor score from baseline was durable at 1 year (53%; 8.9 ± 4.8; 70 patients) and at 2 years (56%; 8.8 ± 5.0; 67 patients). Similarly, the disability score at baseline (16.4 ± 4.5; 76 patients) improved by 64% at 6 months (5.4 ± 4.7; 75 patients). This improvement was also sustained at 1 year (5.4 ± 5.3; 70 patients) and at 2 years (6.5 ± 5.0; 67 patients). Paresthesias and gait disturbances were the most common adverse effects at 1 year-each observed in 10 patients with an additional 5 patients experiencing neurological adverse effects. None of the adverse events worsened over the period of follow-up, and 2 of these resolved. There were no new delayed complications at 2 years. INTERPRETATION Tremor suppression after MRgFUS thalamotomy for ET is stably maintained at 2 years. Latent or delayed complications do not develop after treatment. Ann Neurol 2018;83:107-114.

Therapeutic ultrasound is only beginning to be applied to neurologic conditions, but the potential of this modality for a wide spectrum of brain applications is high. Engineering advances now allow sound waves to be targeted through the skull to a brain region selected with real time magnetic resonance imaging and thermography, using a commercial array of focused emitters. High intensities of sonic energy can create a coagulation lesion similar to that of older radiofrequency stereotactic methods, but without opening the skull. This has led to the recent Food and Drug Administration approval of focused ultrasound (FUS) thalamotomy for unilateral treatment of essential tremor. Clinical studies of stereotactic FUS for aspects of Parkinson’s disease, chronic pain, and refractory psychiatric indications are underway, with promising results. Moderate-intensity FUS has the potential to safely open the blood–brain barrier for localized delivery of therapeutics, while low levels of sonic energy can be used as a form of neuromodulation.

Background Magnetic resonance-guided focused ultrasound ablation of the thalamic ventral intermediate nucleus is a safe and effective treatment for medically refractory essential tremor. However, indirect targeting of the ventral intermediate nucleus using stereotactic coordinates from normal neuroanatomy can be inefficient. We therefore evaluated the feasibility of supplementing this method with direct targeting of the dentato-rubro-thalamic tract. Methods We retrospectively identified four patients undergoing magnetic resonance-guided focused ultrasound ablation for essential tremor in which preoperative diffusion tractography imaging of the dentato-rubro-thalamic tract was fused with T2 weighted-imaging and utilized for intra-procedural targeting. The size and location of the dentato-rubro-thalamic tract and 24-hour lesion, as well as the center of the stereotactic coordinates, was evaluated. Finally, the amount of overlap between the dentato-rubro-thalamic tract and the lesion was calculated. Results The 24-hour lesion size was homogeneous in the cohort (mean 31.3 mm2, range 30–32 mm2), while there was substantial variation in the dentato-rubro-thalamic tract area (mean 14.3 mm2, range 3–24 mm2). The center of the stereotactic coordinates and dentato-rubro-thalamic tract diverged by more than 1 mm in mediolateral and anterposterior directions in all patients, while the dentato-rubro-thalamic tract and lesion centers were in close proximity (mean mediolateral separation 1 mm, range 0.1–2.2 mm; mean anteroposterior separation 0.75 mm, range 0.4–1.2 mm). There was greater than 50% coverage of the dentato-rubro-thalamic tract by the lesion in all patients (mean 82.9%, range 66.7–100%). All patients experienced durable tremor relief. Conclusion Direct targeting of the dentato-rubro-thalamic tract using diffusion tractography imaging fused to T2 weighted-imaging may be a useful strategy for focused ultrasound treatment of essential tremor. Further investigation of the technique is warranted.

This is the first report of the synthesis of a new nanoparticle, sans iron oxide rhodamine B (SIRB), an example of a new class of nanoparticles. SIRB is designed to provide all of the cell labeling properties of the ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle Molday ION Rhodamine B (MIRB) without containing the iron oxide core. MIRB was developed to label cells and allow them to be tracked by MRI or to be manipulated by magnetic gradients. SIRB possesses a similar size, charge and cross-linked dextran coating as MIRB. Of great interest is understanding the biological and physiological changes in cells after they are labeled with a USPIO. Whether these effects are due to the iron oxide buried within the nanoparticle or to the surface coating surrounding the iron oxide core has not been considered previously. MIRB and SIRB represent an ideal pairing of nanoparticles to identify nanoparticle anatomy responsible for post-labeling cytotoxicity. Here we report the effects of SIRB labeling on the SH-SY5Y neuroblastoma cell line and primary human neuroprogenitor cells (hNPCs). These effects are contrasted with the effects of labeling SH-SY5Y cells and hNPCs with MIRB. We find that SIRB labeling, like MIRB labeling, (i) occurs without the use of transfection reagents, (ii) is packaged within lysosomes distributed within cell cytoplasm, (iii) is retained within cells with no loss of label after cell storage, and (iv) does not alter cellular viability or proliferation, and (v) SIRB labeled hNPCs differentiate normally into neurons or astrocytes. Copyright © 2016 John Wiley & Sons, Ltd.

Background Magnetic resonance-guided focused ultrasound thalamotomy (FUS-T) is an emerging treatment for essential tremor (ET). Objective To determine the predictors of outcomes after FUS-T. Methods Two treatment groups were analyzed: 75 ET patients enrolled in the pivotal trial, between 2013 and 2015; and 114 patients enrolled in the postpivotal trials, between 2015 and 2016. All patients had medication-refractory, disabling ET, and underwent unilateral FUS-T. The primary outcome (hand tremor score, 32-point scale with higher scores indicating worse tremor) and the secondary outcome variables (Clinical Rating Scale for Tremor Part C score: 32-point scale with higher scores indicating more disability) were assessed at baseline and 1, 3, 6, and 12 mo. The operative outcome variables (ie, peak temperature, number of sonications) were analyzed. The results between the 2 treatment groups, pivotal and postpivotal, were compared with repeated measures analysis of variance and adjusted for confounding variables. Results A total of 179 patients completed the 12-mo evaluation. The significant predictors of tremor outcomes were patient age, disease duration, peak temperature, and number of sonications. A greater improvement in hand tremor scores was observed in the postpivotal group at all time points, including 12 mo (61.9% ± 24.9% vs 52.1% ± 24.9%, P = .009). In the postpivotal group, higher energy was used, resulting in higher peak temperatures (56.7 ± 2.5 vs 55.6 ± 2.8°C, P = .004). After adjusting for age, years of disease, number of sonications, and maximum temperature, the treatment group was a significant predictor of outcomes (F = 7.9 [1,165], P = .005). Conclusion We observed an improvement in outcomes in the postpivotal group compared to the pivotal group potentially reflecting a learning curve with FUS-T. The other associations of tremor outcomes included patient age, disease duration, peak temperature, and number of sonications.

We propose that magnetic targeting of super-paramagnetic iron oxide nanoparticles (SPION) labeled cells will enhance the delivery of stem cells to brain after focused ultrasound (FUS) mediated opening of the blood-brain barrier (BBB). FUS mediated opening of the BBB allowing stem cells to enter the brain from the blood is a remarkable advance in delivery of stem cell therapeutics over current invasive methods of brain injection. However the efficiency of cellular entry is low. SPIONs are taken up by stem cells, allowing for labeling of transplanted stem cells in brain both with histology and MRI. Our research shows that SPION labeled stem cells show enhanced brain retention near a magnet on the skull, in a rat model of traumatic brain injury. There is no experience combining these two minimally invasive strategies to deliver stem cells to the brain. We will assess the capacity of an external magnet to enhance the efficiency of delivery to brain of SPION loaded stem cells after transient opening of the BBB using FUS. We will evaluate SPION loaded neural stem cells delivered by intravenous infusion in rats that have undergone MRI targeted FUS opening of the BBB along with a magnet placed over the skull of the sonicated hemisphere, to animals with FUS alone. The number and distribution of stem cells will be quantitative for each group to assess enhancement of delivery of stem cells after BBB opening using FUS by the addition of SPION loading and an external magnet.

Background Adult bone marrow contains stem cells that replenish the myeloid and lymphoid lineages. A subset of human and mouse CD34+ bone marrow stem cells can be propagated in culture to autonomously express embryonic stem cell genes and embryonic germ layer lineage genes. The current study was undertaken to determine whether these CD34+ stem cells could be obtained from human blood, whether gene expression could be modulated by culture conditions and whether the cells produce insulin. Methods Human peripheral blood buffy coat cells and mobilized CD34+ cells from human blood and from blood from C57Bl/6 J mice were cultured in hybridoma medium or neural stem cell induction medium supplemented with interleukin (IL)-3, IL-6, and stem cell factor (SCF). Changes in mRNA and protein expression were assessed by Western blot analysis and by immunohistochemistry. Mass spectrometry was used to assess insulin production. Results We were able to culture CD34+ cells expressing embryonic stem cell and embryonic germ layer lineage genes from adult human peripheral blood after standard mobilization procedures and from mouse peripheral blood. Gene expression could be modulated by culture conditions, and the cells produced insulin in culture. Conclusion These results suggest a practical method for obtaining large numbers of CD34+ cells from humans to allow studies on their potential to differentiate into other cell types. Electronic supplementary material The online version of this article (10.1186/s13287-018-0858-5) contains supplementary material, which is available to authorized users.

Magnetic resonance imaging-guided focused ultrasound thalamotomy is approved by the U.S. Food and Drug Administration for treatment of essential tremor. Although this incisionless technology creates an ablative lesion, it potentially avoids serious complications of open stereotactic surgery.To determine the safety profile of magnetic resonance imaging-guided focused ultrasound unilateral thalamotomy for essential tremor, including frequency, and severity of adverse events, including serious adverse events.Analysis of safety data for magnetic resonance imaging-guided focused ultrasound thalamotomy (186 patients, five studies).Procedure-related serious adverse events were very infrequent (1.6%), without intracerebral hemorrhages or infections. Adverse events were usually transient and were commonly rated as mild (79%) and rarely severe (1%). As previously reported, abnormalities in sensation and balance were the commonest thalamotomy-related adverse events.The overall safety profile of magnetic resonance imaging-guided focused ultrasound thalamotomy supports its role as a new option for patients with medically refractory essential tremor. © 2018 International Parkinson and Movement Disorder Society.

To assess concordance between physician assessment and patient-reported symptoms when screening for depression in Parkinson disease (dPD).Depression in Parkinson disease is highly prevalent (∼40%) and has a significant impact on quality of life and disability, yet physician recognition and treatment remain inadequate.One thousand seventy-six patients with PD completed the Brief Symptom Inventory-18 (BSI-18), a screening questionnaire for psychiatric symptoms, which was compared to item #3 (depression) on the Unified Parkinson's Disease Rating Scale (UPDRS).The mean BSI-18 depression score was 51.4 (9.7). Of the 170 (16%) patients screening positive for dPD on the BSI-18, 104 (61%) were not recognized as depressed by neurologists on the UPDRS. Factors associated with lower neurologist recognition included male gender, better mental health quality of life, and lack of antidepressant use.More than 60% of patients screening positive for depression on self-report were not recognized by neurologists on the UPDRS. A patient-reported screening tool for depression may improve recognition and management of dPD.

Magnetic resonance guided focused ultrasound (MRgFUS) has recently been investigated as a new treatment modality for essential tremor (ET), but the durability of the procedure has not yet been evaluated. This study reports results at a 2- year follow-up after MRgFUS thalamotomy for ET.A total of 76 patients with moderate-to-severe ET, who had not responded to at least two trials of medical therapy, were enrolled in the original randomized study of unilateral thalamotomy and evaluated using the clinical rating scale for tremor. Sixty-seven of the patients continued in the open-label extension phase of the study with monitoring for 2 years. Nine patients were excluded by 2 years, for example, because of alternative therapy such as deep brain stimulation (n = 3) or inadequate thermal lesioning (n = 1). However, all patients in each follow-up period were analyzed.Mean hand tremor score at baseline (19.8 ± 4.9; 76 patients) improved by 55% at 6 months (8.6 ± 4.5; 75 patients). The improvement in tremor score from baseline was durable at 1 year (53%; 8.9 ± 4.8; 70 patients) and at 2 years (56%; 8.8 ± 5.0; 67 patients). Similarly, the disability score at baseline (16.4 ± 4.5; 76 patients) improved by 64% at 6 months (5.4 ± 4.7; 75 patients). This improvement was also sustained at 1 year (5.4 ± 5.3; 70 patients) and at 2 years (6.5 ± 5.0; 67 patients). Paresthesias and gait disturbances were the most common adverse effects at 1 year-each observed in 10 patients with an additional 5 patients experiencing neurological adverse effects. None of the adverse events worsened over the period of follow-up, and 2 of these resolved. There were no new delayed complications at 2 years.Tremor suppression after MRgFUS thalamotomy for ET is stably maintained at 2 years. Latent or delayed complications do not develop after treatment. Ann Neurol 2018;83:107-114.

Although the use of ultrasound as a potential therapeutic modality in the brain has been under study for several decades, relatively few neuroscientists or neurologists are familiar with this technology. Stereotactic brain lesioning had been widely used as a treatment for medically refractory patients with essential tremor (ET), Parkinson disease (PD), and dystonia but has been largely replaced by deep brain stimulation (DBS) surgery, with advantages both in safety and efficacy. However, DBS is associated with complications including intracerebral hemorrhage, infection, and hardware malfunction. The occurrence of these complications has spurred interest in less invasive stereotactic brain lesioning methods including magnetic resonance imaging–guided high intensity–focused ultrasound (FUS) surgery. Engineering advances now allow sound waves to be targeted noninvasively through the skull to a brain target. High intensities of sonic energy can create a coagulation lesion similar to that of older radiofrequency stereotactic methods, but without opening the skull, recent Food and Drug Administration approval of unilateral thalamotomy for treatment of ET. Clinical studies of stereotactic FUS for aspects of PD are underway. Moderate intensity, pulsed FUS has also demonstrated the potential to safely open the blood-brain barrier for localized delivery of therapeutics including proteins, genes, and cell-based therapy for PD and related disorders. The goal of this review is to provide basic and clinical neuroscientists with a level of understanding to interact with medical physicists, biomedical engineers, and radiologists to accelerate the application of this powerful technology to brain disease

Introduction: We examined the possibility that tetanus toxin can prevent muscle atrophy associated with limb immobility in rats. Methods: While the knee and ankle joints were immobilized unilaterally, the tibialis anterior (TA) muscle on the immobilized side was injected with 1 μl saline or with 1 ng tetanus toxin. After 2 weeks, TA wet weights, contractile forces, and myofiber sizes from the immobilized sides were compared with those from body weight–matched normal animals. Results: Saline group wet weights decreased and produced less absolute twitch and tetanic force and normalized tetanic force compared with the toxin or normal groups. Cross-sectional areas of saline group type I, IIa, and IId myofibers, and the masses of saline group IIa, IId, IIb, and toxin group IIb myofibers, were smaller compared with the normal group. Conclusions: Tetanus toxin prevented common signs of muscle atrophy and may become a useful adjunct to current rehabilitation strategies. Muscle Nerve 50: 759–766, 2014

Transcranial MRI-guided focused ultrasound (tcMRgFUS) is increasingly used to non-invasively treat a wide variety of neurological disorders including essential tremors, Parkinson's disease, and neuropathic pain. Although this treatment is an MRI-guided procedure, the current pre-treatment screening and planning involve a CT of the head to obtain 3D skull images. These images are necessary for estimating the proportion of absorbed energy and the acoustic phase shift associated with the skull and determining the transmit energy of ultrasonic waves to create thermal lesions at a desired focal spot. Ultrashort echo time (UTE) MR sequences can capture signals from tissues such as bone which have a very short transverse relaxation time. In this manuscript, we assess the use of a UTE based sequence to image the calvarium and test the feasibility of obviating the need for CT based imaging during an MR-guided focused ultrasound therapy. We demonstrate that the segmentation of bone using UTE images leads to similar skull density ratio values as determined from CT with high correlation (r = 0.88; p

The unique ways in which tetanus toxin is handled by the nervous system and its enzymatic activity account for its ability to selectively increase motor neuron activity. At the neuromuscular junction, tetanus toxin binds to motor neuron terminals, which are enriched with receptors for the toxin.1 The toxin is then internalized into membrane-bound endosomes, which are trafficked by retrograde fast axonal transport to the motor neuron cell body in the spinal cord. Once the endosomes reach the cell body, they likely fuse with the plasma membrane, resulting in release of the toxin into the extracellular space, where it binds to presynaptic terminals in the vicinity of motor neurons.2 The toxin then undergoes a second round of endocytosis followed by liberation of the proteolytic light chain portion into the cytosol of the second-order presynaptic terminals. Within the presynaptic terminals of spinal interneurons, the light chain cleaves vesicle associated membrane protein (predominantly the VAMP2 isoform), blocking fusion of synaptic vesicles.3 The result is failure of neurotransmitter release, primarily at inhibitory synapses in the vicinity of motor neurons. The degree of toxin spread from the site of its elaboration likely accounts for whether many muscles, or just one muscle, become affected. Widespread intoxication through the systemic circulation results in the continuous involuntary muscle contractions that characterize clinical generalized tetanus, while spatially restricted uptake of the toxin results in a localized state of muscle hyperexcitability. Tetanus toxin produces repetitive segmental motor neuron discharges in response to muscle stretch.4,5 The resulting increase in limb rigidity is suggestive of some form of reflex hyperactivity, but there is conflicting evidence regarding the effect of the toxin on the stretch reflex. Early as well as more recent reports indicated that the toxin leaves the reflex unchanged,6–9 enhanced,4,5,10,11 or depressed.12 In part, the different outcomes can be attributed to a broad range of toxin dosages, injection locations, and timing of observations. This study used the Hoffman reflex, or “H-reflex,” to examine aspects of the muscle stretch reflex that may be affected by tetanus toxin. The neural pathway that produces the H-reflex does not rely on active inputs from stretch receptors. Consequently the H-reflex offers an incomplete view of the active circuit that controls the stretch reflex. Nonetheless, the H-reflex has become a useful tool in assessing spinal motor reflex function. Six features of H-waves from animals with unilateral hind limb tetanus were examined by peripheral nerve stimulation: (i) threshold, (ii) maximal wave amplitude, (iii) latency, (iv) duration, (v) gain (estimated from the slopes of recruitment curves), and (vi) rate-dependent depression. To examine whether tetanus toxin can modulate the effects of descending inputs to the local stretch reflex, we also studied depression of H-reflex amplitude using a twin pulse paradigm where conditioning activation of descending pathways by magnetic stimulation of the brain/upper spinal cord was followed by a test pulse delivered with conventional electrodes to a peripheral nerve.

Protein delivery vectors can be grouped into two classes, those with specific membrane receptors undergoing conventional endocytosis and cell penetrating peptides (CPP) that have the capacity to cross cell or endosomal membranes. For both forms of vectors, translocation across a membrane is usually an inefficient process. In the current study, a novel vector combining the widely used CPP, Tat and the non-toxic neuronal binding domain of tetanus toxin (fragment C or TTC) was assessed for its capacity to deliver GFP as a test cargo protein to human neural progenitor cells (NPCs). These two functional membrane interacting domains dramatically enhanced internalization of the conjugated cargo protein. Tat-TTC-GFP was found to be bound or internalized at least 83-fold more than Tat-GFP and 33-fold more than TTC-GFP in NPCs by direct fluorimetry, and showed enhanced internalization by quantitative microscopy of 18 - and 14-fold, respectively. This preferential internalization was observed to be specific to neuronal cell types. Photochemical internalization (PCI) was utilized to facilitate escape of the endosome-sequestered proteins. The combined use of the Tat-TTC delivery vector with PCI led to both enhancement of neural cell type specific delivery to an endosomal target, followed by the option of efficient release to the cytosol.

Leucine-rich repeat kinase 2 is a large protein with implications in genetic and sporadic causes of Parkinson's disease. The physiological functions of LRRK2 are largely unknown. In this report, we investigated whether LRRK2 alters neural transport using live-cell imaging techniques and human neuroblastoma SH-SY5Y cells. Our results demonstrated that expression of the PD-linked mutant, LRRK2-R1441C, induced mitochondrial, and lysosomal transport defects in neurites of SH-SY5Y cells. Most importantly, recently identified GTP-binding inhibitors, 68 and FX2149, can reduce LRRK2 GTP-binding activity and attenuates R1441C-induced mitochondrial and lysosomal transport impairments. These results provide direct evidence and an early mechanism for neurite injury underlying LRRK2-induced neurodegeneration. This is the first report to show that LRRK2 GTP-binding activity plays a critical role during neurite transport, suggesting inhibition of LRRK2 GTP-binding could be a potential novel strategy for PD intervention.

MRI guided focused ultrasound surgery (MRgFUS) has been FDA approved for unilateral treatment of essential tremor (ET). Before this non-incisional lesioning method can be applied to the treatment of both hemispheres the previous experience with bilateral thalamic ablation must be addressed. In particular, the high incidence of worsening of speech and balance associated with bilateral surgical thalamotomy, a rationale for the development of deep brain stimulation. The highest incidence of these complication occurred in the early years of surgery for movement disorders, when neither MRI nor current stereotactic methods were available. The vast majority of these initial patients suffering these complications had Parkinson's disease where approximately 30% developed worsening dysarthria and ataxia after bilateral thalamotomy. Patients suffering these complications commonly had baseline abnormalities in speech and balance or worsening symptoms after a first unilateral procedure. The more contemporary experience with bilateral thalamotomy in the ET population is both much more limited in patient numbers (includes patients after Gamma Knife radiosurgery), and shows a much lower rate of these complications (approximately 5%). This more recent experience suggests that bilateral thalamotomy using closed incisionless methods such as MRgFUS has the potential to safely improve ET patients with axial or bilateral limb involvement, if done in a staged manner excluding patients with baseline dysarthria or ataxia or transient worsening of these symptoms following a unilateral procedure.

Uncontrolled pilot studies have suggested the efficacy of focused ultrasound thalamotomy with magnetic resonance imaging (MRI) guidance for the treatment of essential tremor.We enrolled patients with moderate-to-severe essential tremor that had not responded to at least two trials of medical therapy and randomly assigned them in a 3:1 ratio to undergo unilateral focused ultrasound thalamotomy or a sham procedure. The Clinical Rating Scale for Tremor and the Quality of Life in Essential Tremor Questionnaire were administered at baseline and at 1, 3, 6, and 12 months. Tremor assessments were videotaped and rated by an independent group of neurologists who were unaware of the treatment assignments. The primary outcome was the between-group difference in the change from baseline to 3 months in hand tremor, rated on a 32-point scale (with higher scores indicating more severe tremor). After 3 months, patients in the sham-procedure group could cross over to active treatment (the open-label extension cohort).Seventy-six patients were included in the analysis. Hand-tremor scores improved more after focused ultrasound thalamotomy (from 18.1 points at baseline to 9.6 at 3 months) than after the sham procedure (from 16.0 to 15.8 points); the between-group difference in the mean change was 8.3 points (95% confidence interval [CI], 5.9 to 10.7; P0.001). The improvement in the thalamotomy group was maintained at 12 months (change from baseline, 7.2 points; 95% CI, 6.1 to 8.3). Secondary outcome measures assessing disability and quality of life also improved with active treatment (the blinded thalamotomy cohort)as compared with the sham procedure (P0.001 for both comparisons). Adverse events in the thalamotomy group included gait disturbance in 36% of patients and paresthesias or numbness in 38%; these adverse events persisted at 12 months in 9% and 14% of patients, respectively.MRI-guided focused ultrasound thalamotomy reduced hand tremor in patients with essential tremor. Side effects included sensory and gait disturbances. (Funded by InSightec and others; ClinicalTrials.gov number, NCT01827904.).

To compare the effects of gait impairment without freezing of gait (FOG) versus FOG without gait impairment in Parkinson's disease (PD) on disability and quality of life.FOG is frequently characterized as the major cause of gait-related disability in PD. However, gait impairment may also result from other PD symptoms including slowing, motor asymmetry, gait variability, dystonia or stooped posture.The Unified Parkinson's Disease Rating Scale (UPDRS), Older Americans Resources and Services Disability Scale (OARS) and the SF-12 Health Status Survey were used to evaluate patients with PD. Responses to UPDRS Items #14 (Freezing) and # 29 (Gait) were used to create 4 subgroups: 1) No FOG or gait impairment, 2) FOG, no gait impairment, 3) Gait impairment, no FOG, and 4) Both FOG and gait impairment. Disease severity, disability, and quality of life were compared across the subgroups with ANOVAs, and between subgroups with t-tests.916 PD patients were divided into 4 subgroups based on their gait and freezing score (#1: n = 213, #2: n = 41, #3: n = 323 and #4: n = 339). Total UPDRS progressively increased from Group 1 through Group 4 (1 = 25.2, 2 = 33.7, 3 = 39.2, 4 = 59.2; p0.001). Motor UPDRS also progressively increased (1 = 17.4, 2 = 19.7, 3 = 26.9, 4 = 36.5; p0.0001). Similarly, disability and health-related quality of life progressively increased from Group 1 through Group 4 (Total OARS: 1 = 15.3, 2 = 17.2, 3 = 18.9, 4 = 28.4; p0.001). Group 3 (Gait impairment, no FOG) showed greater disease severity than Group 2 (FOG, no gait impairment; Total and Motor UPDRS; p0.05), but the difference did not reach significance for disability or quality of life.Gait impairment without FOG was associated with greater disease severity than FOG without gait impairment. The combination of gait impairment and FOG was associated with the greatest disease severity and disability. These results show differential effects of diverse features of gait impairment in PD and demonstrate the importance of gait features unrelated to freezing.

Initial cellular uptake of cell penetrating peptide (CPP) linked macromolecules is usually endosomal, with passage from endosome to cytosol a major limitation to efficient delivery. To gain a better understanding of the passage of the CPP-linked proteins, we studied the uptake and localization of CPP-linked proteins that contained two different forms of fluorescent markers, GFP protein and chemically conjugated tetramethylrhodamine, in living cells. Rhodamine labeled TAT-GFP was internalized in multiple cell lines including HEK293, N18-RE-105, hippocampal slices, and human neural progenitor cells and showed predominantly endosomal localization of both fluorescent markers. Cytosolic localization of some rhodamine label was detected to suggest that some of the GFP label had exited from the endosome. However, quantification of the distribution of the rhodamine and GFP label indicated that the protein location was primarily endosomal and that the distribution of TAT-GFP was not significantly different than that of an exclusively endosomal localized exogenous protein (tetanus toxin fragment C - TTC). As a result, photochemical internalization (PCI) was evaluated and caused a significant quantitative redistribution of cellular fluorescence of rhodamine and GFP labels to demonstrate increased cytosolic delivery of GFP. While rhodamine-labeled TAT-GFP showed cytosolic delivery with exposure to specific wavelengths of fluorescent illumination, a similarly labeled GFP fusion protein containing the membrane binding domain of TTC did not mediate PCI in N18-RE-105 cells.

Botulinum neurotoxins (BoNTs) are the most potent natural toxins known. The effects of BoNT serotype A (BoNT/A) can last several months, whereas the effects of BoNT serotype E (BoNT/E), which shares the same synaptic target, synaptosomal-associated protein 25 (SNAP25), last only several weeks. The long-lasting effects or persistence of BoNT/A, although desirable for therapeutic applications, presents a challenge for medical treatment of BoNT intoxication. Although the mechanisms for BoNT toxicity are well known, little is known about the mechanisms that govern the persistence of the toxins. We show that the recombinant catalytic light chain (LC) of BoNT/E is ubiquitylated and rapidly degraded in cells. In contrast, BoNT/A LC is considerably more stable. Differential susceptibility of the catalytic LCs to ubiquitin-dependent proteolysis therefore might explain the differential persistence of BoNT serotypes. In this regard we show that TRAF2, a RING finger protein implicated in ubiquitylation, selectively associates with BoNT/E LC and promotes its proteasomal degradation. Given these data, we asked whether BoNT/A LC could be targeted for rapid proteasomal degradation by redirecting it to characterized ubiquitin ligase domains. We describe chimeric SNAP25-based ubiquitin ligases that target BoNT/A LC for degradation, reducing its duration in a cellular model for toxin persistence.

Exposure to a number of drugs, chemicals, or environmental factors can cause parkinsonism. Epidemiologic evidence supports a causal link between the consumption of flour made from the washed seeds of the plant Cycas micronesica by the Chamorro population of Guam and the development of amyotrophic lateral sclerosis/parkinsonism dementia complex.We now report that consumption of washed cycad flour pellets by Sprague-Dawley male rats induces progressive parkinsonism.Cycad-fed rats displayed motor abnormalities after 2 to 3 months of feeding such as spontaneous unilateral rotation, shuffling gait, and stereotypy. Histological and biochemical examination of brains from cycad-fed rats revealed an initial decrease in the levels of dopamine and its metabolites in the striatum (STR), followed by neurodegeneration of dopaminergic (DAergic) cell bodies in the substantia nigra (SN) pars compacta (SNc). alpha-Synuclein (alpha-syn; proteinase K-resistant) and ubiquitin aggregates were found in the DAergic neurons of the SNc and neurites in the STR. In addition, we identified alpha-syn aggregates in neurons of the locus coeruleus and cingulate cortex. No loss of motor neurons in the spinal cord was found after chronic consumption of cycad flour. In an organotypic slice culture of the rat SN and the striatum, an organic extract of cycad causes a selective loss of dopamine neurons and alpha-syn aggregates in the SN.Cycad-fed rats exhibit progressive behavioral, biochemical, and histological hallmarks of parkinsonism, coupled with a lack of fatality.

With a view toward improving delivery of exogenous glial cell line-derived neurotrophic factor (GDNF) to CNS motor neurons in vivo, we evaluated the bioavailability and pharmacological activity of a recombinant GDNF:tetanus toxin C-fragment fusion protein in mouse CNS. Following intramuscular injection, GDNF:TTC but not recombinant GDNF (rGDNF) produced strong GDNF immunostaining within ventral horn cells of the spinal cord. Intrathecal infusion of GDNF:TTC resulted in tissue concentrations of GDNF in lumbar spinal cord that were at least 150-fold higher than those in mice treated with rGDNF. While levels of immunoreactive choline acetyltransferase and GFRalpha-1 in lumbar cord were not altered significantly by intrathecal infusion of rGNDF, GDNF:TTC, or TTC, only rGDNF and GDNF:TTC caused significant weight loss following intracerebroventricular infusion. These studies indicate that insect cell-derived GDNF:TTC retains its bi-functional activity in mammalian CNS in vivo and improves delivery of GDNF to spinal cord following intramuscular- or intrathecal administration.

The capacity of tetanus toxin to enhance motor neuron excitability has suggested its potential use as a therapeutic. Widespread active vaccination against tetanus in all developed countries is considered the major obstacle to clinical use of the toxin. We wished to determine the response to localized intramuscular injection of tetanus toxin in both passively and actively immunized animals as an initial exploration into the possible use of tetanus toxin as a clinical therapeutic. Unvaccinated mice (n=18) underwent intramuscular injection of tetanus toxin into the gastrocnemius muscle (0.2 ng, 1 ng, 5 ng). All animals in the lowest dose group developed only local tetanus of the injected limb of at least 2 weeks duration, while all animals in the higher dose groups also rapidly developed generalized tetanus and were euthanized. Another group of mice (n=20) received anti-tetanus immunoglobulin (20-40 IU) at the time of toxin injection. These animals although dramatically resistant to the toxin developed predominantly local tetanus for over one month at doses of 2.5 microg and 5.0 microg. A third group of mice (n=30) underwent active vaccination with tetanus toxoid to induce protective anti-tetanus immunity and then was challenged with high dose toxin injection (5 ng, 50 ng, 0.5 microg, 1.25 microg, 2.5 microg, or 5 microg). All animals developed local tetanus in the injected limb at a dose of at least 0.5 microg. The severity and duration of local tetanus was generally related to dose, but was more variable in the actively vaccinated group than in the naive or passively immunized animals. Response to the toxin over the first few days was predictive of both the duration and maximal severity of the motor response. Although vaccination dramatically increases resistance to tetanus toxin, by virtue of its extremely high potency, the toxin can produce prolonged localized tetanus even in vaccinated animals with relatively small amounts of protein. These results suggest the possible use of tetanus toxin to enhance local motor activity in a variety of neurologic conditions even in immunized humans. This study in uniformly vaccinated animals also illustrates the potential difficulties in determining an appropriate dose of toxin in a human population with variable degrees of immunity.

The objective of this study is to compare physical performance measures for their ability to discriminate between levels of disability and disease severity in Parkinson's disease (PD). Disability in PD is commonly assessed by patient self-report, which may be limited by patient insight. Methods: Seventy-nine patients with PD were tested with seven performance measures: Physical Performance Test (PPT), modified Physical Performance Test (mPPT), Short Physical Performance Battery (SPPB), Performance Test of Activities of Daily Living (PADL), Berg Balance Scale (BBS), Timed Up and Go (TUG), and Functional Reach (FR). These measures were compared with patient-reported disability on the Older Americans Resource and Services Disability subscale (OARS) and disease severity on the Unified Parkinson's Disease Rating Scale (UPDRS). The performance measures were more sensitive to levels of disease severity than disability. Four measures discriminated across quartiles of disability (PPT, mPPT, BBS, TUG: P < 0.05), whereas all seven measures discriminated across quartiles of the Total UPDRS (PPT, mPPT, BBS, TUG, FR: P < 0.01; SPPB, PADL: P < 0.05). However, no measure consistently discriminated between subgroups with a range of early and advanced disease severity. The seven physical performance measures showed different profiles of strengths and weaknesses in assessing disability and disease severity. The results of this study will facilitate choosing performance measures for clinical care and clinical trials in PD. © 2008 Movement Disorder Society

The objectives of this study are to assess the level of disease severity associated with disability in Parkinson disease (PD) and the sequence of loss of independence in basic and instrumental activities of daily living (ADLs and IADLs). Six hundred eighteen patients with PD were evaluated for disease severity with the Unified PD Rating Scale (UPDRS) and for disability with the Older Americans Resource and Services Disability Subscale (OARS). The association between patient-reported disability on ADLs and IADLs and level of disease severity on the total UPDRS was examined cross-sectionally. Disability, with loss of independent function is reported between total UPDRS scores 30 to 40, and HY stages II to III. Difficulty with daily activities, without loss of independent function is reported earlier, at UPDRS

Although resting tremor is the most identifiable sign of Parkinson's disease, its underlying basis appears to be the most complex of the cardinal signs. The variable relationship of resting tremor to other symptoms of PD has implications for diagnosis, prognosis, medical and surgical treatment. Structural lesions very rarely cause classic resting tremor, with likely contributions to tremor by a network of neurons both within and outside the basal ganglia. Patients with only resting tremor show dopaminergic deficits with radioligand imaging, but severity of tremor correlates poorly in such dopamine imaging studies. Correlation of tremor severity to changes in radioligand studies is also limited by the use of mostly qualitative measures of tremor severity. A complex pharmacologic basis of parkinsonian resting tremor is supported by treatment studies. Although levodopa is clearly effective for resting tremor, several agents have shown efficacy that appears to be superior or additive to that of levodopa including anticholinergics, clozapine, pramipexole, and budipine. Although the thalamus has the greatest body of evidence supporting its role as an effective target for surgical treatment of tremor, recent studies suggest that the subthalamic nucleus may be a reasonable alternative target for patients with Parkinson's disease and severe tremor as the predominant symptom.

Although the majority of agents with antiexcitotoxic action act as glutamate receptor antagonists, enzymatic degradation of glutamate can also be neuroprotective. The very low specific activity of the mammalian form of glutamate decarboxylase (GAD), the enzyme that catalyzes the formation of gamma-aminobutyric acid (GABA) from glutamate in neurons, is likely to limit its utility as an antiglutamate neuroprotectant. In contrast, the bacterial form of GAD can be isolated with relatively high specific activity and is most active in acidic environments. We have expressed and purified GAD from Escherichia coli (bGAD) and tested the ability of the enzyme to protect against glutamate excitotoxicity. Incubation of rat hipppocampal slices with the potassium channel antagonist tetraethyl ammonium (TEA) resulted in widespread excitotoxic death of pyramidal and granule cell neurons. bGAD alone showed no significant neurotoxicity and significantly reduced excitotoxicity induced by TEA. We hypothesize that bGAD may be internalized into the synaptic vesicle compartment by nonspecific endocytosis, where both the appropriate pH and high glutamate concentrations are present. Targeting of this enzyme to the interior of synaptic vesicles may enhance its potency as a neuroprotectant against excitotoxicity.

Patients with psychogenic movement disorders (PMD) often report severe impairment, yet the impact of PMD on disability and quality of life has not been examined. We compared 66 patients with PMD and 704 patients with Parkinson's disease (PD) on measures of disability (Older Americans Resources and Services Scale, OARS); quality of life (QOL; SF-12v2 Health Survey) and psychiatric symptomatology (Brief Symptom Inventory 18, BSI-18). On the total OARS, PMD and PD patients reported similar levels of disability (17.6 +/- 6.6, 19.8 +/- 10.9, P = 0.490 at "best" function and 24.1 +/- 11.2, 26.2 +/- 14.3, P = 0.497 at their "worst" function). PMD patients reported similar Physical Health QOL to PD patients (38.9 +/- 14.5, 39.8 +/- 11.6, P = 0.652) but worse mental health QOL (41.6 +/- 13.4 vs. 48.9 +/- 11.0, P < 0.001). On the BSI-18, PMD patients reported higher levels of distress on the Global Symptom Index (62.03 +/- 9.6 vs. 53.7 +/- 9.9, P < 0.001) and on Anxiety, Depression and Somatization subscales (PMD vs. PD scores: Anxiety 58.9 +/- 12.0 vs. 52.3 +/- 10.1, P < 0.001; Depression 58.8 +/- 11.9 vs. 51.3 +/- 10.3, P < 0.001; Somatization 60.5 +/- 11.0 vs. 54.7 +/- 8.7, P < 0.001). Thus, severity of disability reported by the PMD group was equal to that seen in a progressive neurodegenerative condition. Quality of life and mental health implications of PMD were also evident. PMD impacts several aspects of patient function and daily life.

Although recombinant glucocerebrosidase (GCase) is the standard therapy for the inherited lysosomal storage disease Gaucher's disease (GD), enzyme replacement is not effective when the central nervous system is affected. We created a series of recombinant genes/proteins where GCase was linked to different membrane binding peptides including the Tat peptide, the rabies glycoprotein derived peptide (RDP), the binding domain from tetanus toxin (TTC), and a tetanus like peptide (Tet1). The majority of these proteins were well-expressed in a mammalian producer cell line (HEK 293F). Purified recombinant Tat-GCase and RDP-GCase showed similar GCase protein delivery to a neuronal cell line that genetically lacks the functional enzyme, and greater delivery than control GCase, Cerezyme (Genzyme). This initial result was unexpected based on observations of superior protein delivery to neurons with RDP as a vector. A recombinant protein where a fragment of the flexible hinge region from IgA (IgAh) was introduced between RDP and GCase showed substantially enhanced GCase neuronal delivery (2.5 times over Tat-GCase), suggesting that the original construct resulted in interference with the capacity of RDP to bind neuronal membranes. Extended treatment of these knockout neuronal cells with either Tat-GCase or RDP-IgAh-GCase resulted in an >90% reduction in the lipid substrate glucosylsphingosine, approaching normal levels. Further in vivo studies of RDP-IgAh-GCase as well as Tat-GCase are warranted to assess their potential as treatments for neuronopathic forms of GD. These peptide vectors are especially attractive as they have the potential to carry a protein across the blood-brain barrier, avoiding invasive direct brain delivery.

Stem cell therapy is under active investigation for traumatic brain injury (TBI). Noninvasive stem cell delivery is the preferred method, but retention of stem cells at the site of injury in TBI has proven challenging and impacts effectiveness. To investigate the effects of applying a magnetic field on cell homing and retention, we delivered human neuroprogenitor cells (hNPCs) labeled with a superparamagnetic nanoparticle into post-TBI animals in the presence of a static magnetic field. We have previously devised a method of loading hNPCs with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles Molday ION Rhodamine B (MIRB™). Labeling of hNPCs (MIRB-hNPCs) does not affect hNPC viability, proliferation, or differentiation. The 0.6 tesla (T) permanent magnet was placed ~4 mm above the injured parietal cortex prior to intracarotid injection of 4 × 104 MIRB-hNPCs. Fluorescence imaging, Perls' Prussian blue histochemistry, immunocytochemistry with SC121, a human-specific antibody, and T2-weighted magnetic resonance imaging ex vivo revealed there was increased homing and retention of MIRB-hNPCs in the injured cortex as compared to the control group in which MIRB-hNPCs were injected in the absence of a static magnetic field. Fluoro-Jade C staining and immunolabeling with specific markers confirmed the viability status of MIRB-hNPCs posttransplantation. These results show that increased homing and retention of MIRB-hNPCs post-TBI by applying a static magnetic field is a promising technique to deliver cells into the CNS for treatment of neurological injuries and neurodegenerative diseases.

This is the first report of the synthesis of a new nanoparticle, sans iron oxide rhodamine B (SIRB), an example of a new class of nanoparticles. SIRB is designed to provide all of the cell labeling properties of the ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle Molday ION Rhodamine B (MIRB) without containing the iron oxide core. MIRB was developed to label cells and allow them to be tracked by MRI or to be manipulated by magnetic gradients. SIRB possesses a similar size, charge and cross-linked dextran coating as MIRB. Of great interest is understanding the biological and physiological changes in cells after they are labeled with a USPIO. Whether these effects are due to the iron oxide buried within the nanoparticle or to the surface coating surrounding the iron oxide core has not been considered previously. MIRB and SIRB represent an ideal pairing of nanoparticles to identify nanoparticle anatomy responsible for post-labeling cytotoxicity. Here we report the effects of SIRB labeling on the SH-SY5Y neuroblastoma cell line and primary human neuroprogenitor cells (hNPCs). These effects are contrasted with the effects of labeling SH-SY5Y cells and hNPCs with MIRB. We find that SIRB labeling, like MIRB labeling, (i) occurs without the use of transfection reagents, (ii) is packaged within lysosomes distributed within cell cytoplasm, (iii) is retained within cells with no loss of label after cell storage, and (iv) does not alter cellular viability or proliferation, and (v) SIRB labeled hNPCs differentiate normally into neurons or astrocytes. Copyright © 2016 John WileySons, Ltd.

The purpose of this study is to address the use of movement assessment sensors for clinical diagnosis and treatment. Eleven patients with Parkinson's disease who had under-gone deep brain stimulation (DBS) surgery were observed during follow-up appointments for adjustments to the stimulation settings. We examine the ways in which the patients and clinicians assess movement ability together in the clinic and how these assessments relate to the treatment of functional disability through DBS. We have found that effective assessment of movement and treatment efficacy is a collaborative and interpretive process (co-interpretation) that relies on input from patients, clinicians, and caregivers. From these findings we describe the design directions for movement sensors to support co-interpretation of movement in a clinical context as opposed to simply movement definition.

Background Mitochondrial dysfunction is a hallmark of neurodegenerative diseases including Alzheimer’s disease (AD), with morphological and functional abnormalities limiting the electron transport chain and ATP production. A contributing factor of mitochondrial abnormalities is loss of nicotinamide adenine dinucleotide (NAD), an important cofactor in multiple metabolic reactions. Depletion of mitochondrial and consequently cellular NAD(H) levels by activated NAD glycohydrolases then culminates in bioenergetic failure and cell death. De Novo NAD+ synthesis from tryptophan requires a multi-step enzymatic reaction. Thus, an alternative strategy to maintain cellular NAD+ levels is to administer NAD+ precursors facilitating generation via a salvage pathway. We administered nicotinamide mononucleotide (NMN), an NAD+ precursor to APP(swe)/PS1(ΔE9) double transgenic (AD-Tg) mice to assess amelioration of mitochondrial respiratory deficits. In addition to mitochondrial respiratory function, we examined levels of full-length mutant APP, NAD+-dependent substrates (SIRT1 and CD38) in homogenates and fission/fusion proteins (DRP1, OPA1 and MFN2) in mitochondria isolated from brain. To examine changes in mitochondrial morphology, bigenic mice possessing a fluorescent protein targeted to neuronal mitochondria (CaMK2a-mito/eYFP), were administered NMN. Methods Mitochondrial oxygen consumption rates were examined in N2A neuroblastoma cells and non-synaptic brain mitochondria isolated from mice (3 months). Western blotting was utilized to assess APP, SIRT1, CD38, DRP1, OPA1 and MFN2 in brain of transgenic and non-transgenic mice (3–12 months). Mitochondrial morphology was assessed with confocal microscopy. One-way or two-way analysis of variance (ANOVA) and post-hoc Holm-Sidak method were used for statistical analyses of data. Student t-test was used for direct comparison of two groups. Results We now demonstrate that mitochondrial respiratory function was restored in NMN-treated AD-Tg mice. Levels of SIRT1 and CD38 change with age and NMN treatment. Furthermore, we found a shift in dynamics from fission to fusion proteins in the NMN-treated mice. Conclusions This is the first study to directly examine amelioration of NAD+ catabolism and changes in mitochondrial morphological dynamics in brain utilizing the immediate precursor NMN as a potential therapeutic compound. This might lead to well-defined physiologic abnormalities that can serve an important role in the validation of promising agents such as NMN that target NAD+ catabolism preserving mitochondrial function.

Background: Increasing evidence points toward the efficacy of nutritional modifications in delaying cognitive decline and mood/behavioral difficulties in Alzheimer’s disease (AD). Nutritional supplementation with individual agents has shown varied results suggesting the need for combinatorial intervention. Objective: We set out to determine whether nutritional intervention could positively impact cognitive performance and behavioral difficulties for individuals diagnosed with AD. Methods: A double-blind, multi-site, phase II study (ClinicalTrials.gov NCT01320527; Alzheimer’s Association Trialmatch) was conducted in which 106 individuals with AD were randomized to a nutraceutical formulation (NF; folate, alpha-tocopherol, B12, S-adenosyl methioinine, N-acetyl cysteine, acetyl-L-carnitine) or placebo for 3 or 6 months, followed by an open-label extension where participants received NF for 6 additional months. Results: The NF cohort improved versus the placebo cohort within 3 months (Clox-1 p = 0.0083, 95%CI [0.4481, 2.9343]; Dementia Rating Scale p = 0.0266, 95%CI [0.1722, 2.7171]). Caregivers reported non-significant improvements in Neuropsychiatric Inventory. Both cohorts improved or maintained baseline performance during open-label extensions. Activities of Daily Living did not change for either cohort. Conclusions: These findings extend phase I studies where NF maintained or improved cognitive performance and mood/behavior.

Transcription factors are fate determining regulatory factors in dopaminergic neuronal development and differentiation. Among them, Nurr1 is the most extensively studied, but the importance of Pitx3 has recently been appreciated. Over-expression of both factors has been utilized to enhance the dopaminergic differentiation of stem cells for transplantation into models of Parkinson's disease. Previous studies however have seen conflicting results regarding the induction of tyrosine hydroxylase expression and dopaminergic differentiation induced by over-expression of Pitx3. Here we show that over-expression of Pitx3 and Nurr1 induced endogenous tyrosine hydroxylase expression as well as a tyrosine hydroxylase promoter–reporter construct in a human non-neuronal and mouse embryonic stem cell lines. Combined simultaneous expression of Nurr1 and Pitx3 however did not lead to enhancement of tyrosine hydroxylase expression over that of either factor alone in either of the cell lines or with either method. These results suggest that other regulatory elements may also be involved in regulation of tyrosine hydroxylase expression. There was also a lack of a correlation between the expression levels of tyrosine hydroxylase with that of the transcription factor constructs. To yield a robust dopaminergic differentiation a combinatorial or successive treatment with different transcription factors may be more effective.