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In Parkinson's disease (PD), the second most common neurodegenerative disorder, non-motor symptoms often precede the development of debilitating motor symptoms and present a severe impact on the quality of life. Lewy bodies containing misfolded α-synuclein progressively develop in neurons throughout the peripheral and central nervous system, which may be correlated with the early development of non-motor symptoms. Among those, increased fear and anxiety is frequent in PD and thought to result from pathology outside the dopaminergic system, which has been the focus of symptomatic treatment to alleviate motor symptoms. Alpha-synuclein accumulation has been reported in the amygdala of PD patients, a brain region critically involved in fear and anxiety. Here we asked whether α-synuclein overexpression alone is sufficient to induce an enhanced fear phenotype in vivo and which pathological mechanisms are involved. Transgenic mice expressing human wild-type α-synuclein (Thy1-aSyn), a well-established model of PD, were subjected to fear conditioning followed by extinction and then tested for extinction memory retention followed by histopathological analysis. Thy1-aSyn mice showed enhanced tone fear across acquisition and extinction compared to wild-type littermates, as well as a trend to less retention of fear extinction. Immunohistochemical analysis of the basolateral nucleus of the amygdala, a nucleus critically involved in tone fear learning, revealed extensive α-synuclein pathology, with accumulation, phosphorylation, and aggregation of α-synuclein in transgenic mice. This pathology was accompanied by microgliosis and parvalbumin neuron loss in this nucleus, which could explain the enhanced fear phenotype. Importantly, this non-motor phenotype was detected in the pre-clinical phase, prior to dopamine loss in Thy1-aSyn mice, thus replicating observations in patients. Results obtained in this study suggest a possible mechanism by which increased anxiety and maladaptive fear processing may occur in PD, opening a door for therapeutic options and further early biomarker research., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Primary age-related tauopathy (PART) is a neurodegenerative entity defined as Alzheimer-type neurofibrillary degeneration primarily affecting the medial temporal lobe with minimal to absent amyloid-β (Aβ) plaque deposition. The extent to which PART can be differentiated pathoanatomically from Alzheimer disease (AD) is unclear. Here, we examined the regional distribution of tau pathology in a large cohort of postmortem brains (n = 914). We found an early vulnerability of the CA2 subregion of the hippocampus to neurofibrillary degeneration in PART, and semiquantitative assessment of neurofibrillary degeneration in CA2 was significantly greater than in CA1 in PART. In contrast, subjects harboring intermediate-to-high AD neuropathologic change (ADNC) displayed relative sparing of CA2 until later stages of their disease course. In addition, the CA2/CA1 ratio of neurofibrillary degeneration in PART was significantly higher than in subjects with intermediate-to-high ADNC burden. Furthermore, the distribution of tau pathology in PART diverges from the Braak NFT staging system and Braak stage does not correlate with cognitive function in PART as it does in individuals with intermediate-to-high ADNC. These findings highlight the need for a better understanding of the contribution of PART to cognitive impairment and how neurofibrillary degeneration interacts with Aβ pathology in AD and PART., (© 2020 American Association of Neuropathologists, Inc. All rights reserved.)

The human immunodeficiency virus-1 (HIV-1) envelope protein gp120 is the major contributor to the pathogenesis of HIV-associated neurocognitive disorder (HAND). Neuroinflammation plays a pivotal role in gp120-induced neuropathology, but how gp120 triggers neuroinflammatory processes and subsequent neuronal death remains unknown. Here, we provide evidence that NLRP3 is required for gp120-induced neuroinflammation and neuropathy. Our results showed that gp120-induced NLRP3-dependent pyroptosis and IL-1β production in microglia. Inhibition of microglial NLRP3 inflammasome activation alleviated gp120-mediated neuroinflammatory factor release and neuronal injury. Importantly, we showed that chronic administration of MCC950, a novel selective NLRP3 inhibitor, to gp120 transgenic mice not only attenuated neuroinflammation and neuronal death but also promoted neuronal regeneration and restored the impaired neurocognitive function. In conclusion, our data revealed that the NLRP3 inflammasome is important for gp120-induced neuroinflammation and neuropathology and suggest that NLRP3 is a potential novel target for the treatment of HAND.

α-Synuclein (αSyn) is central to the neuropathology of Parkinson's disease (PD) due to its propensity for misfolding and aggregation into neurotoxic oligomers. Nitration/oxidation of αSyn leads to dityrosine crosslinking and aggregation. Myeloperoxidase (MPO) is an oxidant-generating enzyme implicated in neurodegenerative diseases. In the present work we have examined the impact of MPO in PD through analysis of postmortem PD brain and in a novel animal model in which we crossed a transgenic mouse expressing the human MPO (hMPO) gene to a mouse expressing human αSyn-A53T mutant (A53T) (hMPO-A53T). Surprisingly, our results show that in PD substantia nigra, the hMPO gene is expressed in neurons containing aggregates of nitrated αSyn as well as MPO-generated HOCl-modified epitopes. In our hMPO-A53T mouse model, we also saw hMPO expression in neurons but not mouse MPO. In the mouse model, hMPO was expressed in neurons colocalizing with nitrated αSyn, carbamylated lysine, nitrotyrosine, as well as HOCl-modified epitopes/proteins. RNAscope in situ hybridization confirmed hMPO mRNA expression in neurons. Interestingly, the hMPO protein expressed in hMPO-A53T brain is primarily the precursor proMPO, which enters the secretory pathway potentially resulting in interneuronal transmission of MPO and oxidative species. Importantly, the hMPO-A53T mouse model, when compared to the A53T model, exhibited significant exacerbation of motor impairment on rotating rods, balance beams, and wire hang tests. Further, hMPO expression in the A53T model resulted in earlier onset of end stage paralysis. Interestingly, there was a high concentration of αSyn aggregates in the stratum lacunosum moleculare of hippocampal CA2 region, which has been associated in humans with accumulation of αSyn pathology and neural atrophy in dementia with Lewy bodies. This accumulation of αSyn aggregates in CA2 was associated with markers of endoplasmic reticulum (ER) stress and the unfolded protein response with expression of activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), MPO, and cleaved caspase-3. Together these findings suggest that MPO plays an important role in nitrative and oxidative damage that contributes to αSyn pathology in synucleinopathies., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The maintenance of genomic integrity is essential for normal cellular functions. However, it is difficult to maintain over a lifetime in postmitotic cells such as neurons, in which DNA damage increases with age and is exacerbated by multiple neurological disorders, including Alzheimer's disease (AD). Here we used immunohistochemical staining to detect DNA double strand breaks (DSBs), the most severe form of DNA damage, in postmortem brain tissues from patients with mild cognitive impairment (MCI) or AD and from cognitively unimpaired controls. Immunostaining for γH2AX-a post-translational histone modification that is widely used as a marker of DSBs-revealed increased proportions of γH2AX-labeled neurons and astrocytes in the hippocampus and frontal cortex of MCI and AD patients, as compared to age-matched controls. In contrast to the focal pattern associated with DSBs, some neurons and glia in humans and mice showed diffuse pan-nuclear patterns of γH2AX immunoreactivity. In mouse brains and primary neuronal cultures, such pan-nuclear γH2AX labeling could be elicited by increasing neuronal activity. To assess whether pan-nuclear γH2AX represents DSBs, we used a recently developed technology, DNA damage in situ ligation followed by proximity ligation assay, to detect close associations between γH2AX sites and free DSB ends. This assay revealed no evidence of DSBs in neurons or astrocytes with prominent pan-nuclear γH2AX labeling. These findings suggest that focal, but not pan-nuclear, increases in γH2AX immunoreactivity are associated with DSBs in brain tissue and that these distinct patterns of γH2AX formation may have different causes and consequences. We conclude that AD is associated with an accumulation of DSBs in vulnerable neuronal and glial cell populations from early stages onward. Because of the severe adverse effects this type of DNA damage can have on gene expression, chromatin stability and cellular functions, DSBs could be an important causal driver of neurodegeneration and cognitive decline in this disease.

Despite the advent of highly active anti-retroviral therapy HIV-associated neurocognitive disorders (HAND) continue to be a significant problem. Furthermore, the precise pathogenesis of this neurodegeneration is still unclear. The objective of this study was to examine the relationship between infection by the simian immunodeficiency virus (SIV) and neuronal injury in the rhesus macaque using in vivo and postmortem sampling techniques. The effect of SIV infection in 23 adult rhesus macaques was investigated using an accelerated NeuroAIDS model. Disease progression was modulated either with combination anti-retroviral therapy (cART, 4 animals) or minocycline (7 animals). Twelve animals remained untreated. Viral loads were monitored in the blood and cerebral spinal fluid, as were levels of activated monocytes in the blood. Neuronal injury was monitored in vivo using magnetic resonance spectroscopy. Viral RNA was quantified in brain tissue of each animal postmortem using reverse transcription polymerase chain reaction (RT-PCR), and neuronal injury was assessed by immunohistochemistry. Without treatment, viral RNA in plasma, cerebral spinal fluid, and brain tissue appears to reach a plateau. Neuronal injury was highly correlated both to plasma viral levels and a subset of infected/activated monocytes (CD14+CD16+), which are known to traffic the virus into the brain. Treatment with either cART or minocycline decreased brain viral levels and partially reversed alterations in in vivo and immunohistochemical markers for neuronal injury. These findings suggest there is significant turnover of replicating virus within the brain and the severity of neuronal injury is directly related to the brain viral load.

Multiple system atrophy (MSA) is a fatal neurodegenerative disorder characterized by the abnormal accumulation of toxic forms of the synaptic protein alpha-synuclein (α-syn) within oligodendrocytes and neurons. The presence of α-syn within oligodendrocytes in the form of glial cytoplasmic inclusions is the diagnostic hallmark of MSA. However, it has been postulated that α-syn is produced in neurons and propagates to oligodendrocytes, where unknown mechanisms lead to its accumulation. The presence of α-syn within neurons in MSA has not been so extensively studied, but it may shed light into neuropathological mechanisms leading to oligodendroglial accumulation. Here we summarize the principal neuropathological events of MSA, and discuss how a deeper knowledge of these mechanisms may help develop effective therapies targeting α-syn accumulation and spreading., (Copyright © 2017. Published by Elsevier B.V.)

The autophagy-lysosome pathway (ALP) regulates intracellular homeostasis of the cytosolic protein SNCA/alpha-synuclein and is impaired in synucleinopathies, including Parkinson disease and dementia with Lewy bodies (DLB). Emerging evidence suggests that ALP influences SNCA release, but the underlying cellular mechanisms are not well understood. Several studies identified SNCA in exosome/extracellular vesicle (EV) fractions. EVs are generated in the multivesicular body compartment and either released upon its fusion with the plasma membrane, or cleared via the ALP. We therefore hypothesized that inhibiting ALP clearance 1) enhances SNCA release via EVs by increasing extracellular shuttling of multivesicular body contents, 2) alters EV biochemical profile, and 3) promotes SNCA cell-to-cell transfer. Indeed, ALP inhibition increased the ratio of extra- to intracellular SNCA and upregulated SNCA association with EVs in neuronal cells. Ultrastructural analysis revealed a widespread, fused multivesicular body-autophagosome compartment. Biochemical characterization revealed the presence of autophagosome-related proteins, such as LC3-II and SQSTM1. This distinct "autophagosome-exosome-like" profile was also identified in human cerebrospinal fluid (CSF) EVs. After a single intracortical injection of SNCA-containing EVs derived from CSF into mice, human SNCA colocalized with endosome and neuronal markers. Prominent SNCA immunoreactivity and a higher number of neuronal SNCA inclusions were observed after DLB patient CSF EV injections. In summary, this study provides compelling evidence that a) ALP inhibition increases SNCA in neuronal EVs, b) distinct ALP components are present in EVs, and c) CSF EVs transfer SNCA from cell to cell in vivo. Thus, macroautophagy/autophagy may regulate EV protein composition and consequently progression in synucleinopathies.

The computational properties of the human brain arise from an intricate interplay between billions of neurons connected in complex networks. However, our ability to study these networks in healthy human brain is limited by the necessity to use non-invasive technologies. This is in contrast to animal models where a rich, detailed view of cellular-level brain function with cell-type-specific molecular identity has become available due to recent advances in microscopic optical imaging and genetics. Thus, a central challenge facing neuroscience today is leveraging these mechanistic insights from animal studies to accurately draw physiological inferences from non-invasive signals in humans. On the essential path towards this goal is the development of a detailed 'bottom-up' forward model bridging neuronal activity at the level of cell-type-specific populations to non-invasive imaging signals. The general idea is that specific neuronal cell types have identifiable signatures in the way they drive changes in cerebral blood flow, cerebral metabolic rate of O2 (measurable with quantitative functional Magnetic Resonance Imaging), and electrical currents/potentials (measurable with magneto/electroencephalography). This forward model would then provide the 'ground truth' for the development of new tools for tackling the inverse problem-estimation of neuronal activity from multimodal non-invasive imaging data.This article is part of the themed issue 'Interpreting BOLD: a dialogue between cognitive and cellular neuroscience'., (© 2016 The Author(s).)

Unlabelled: Alzheimer's disease (AD) is characterized by the progressive accumulation of amyloid β (Aβ) and microtubule associate protein tau, leading to the selective degeneration of neurons in the neocortex, limbic system, and nucleus basalis, among others. Recent studies have shown that α-synuclein (α-syn) also accumulates in the brains of patients with AD and interacts with Aβ and tau, forming toxic hetero-oligomers. Although the involvement of α-syn has been investigated extensively in Lewy body disease, less is known about the role of this synaptic protein in AD. Here, we found that reducing endogenous α-syn in an APP transgenic mouse model of AD prevented the degeneration of cholinergic neurons, ameliorated corresponding deficits, and recovered the levels of Rab3a and Rab5 proteins involved in intracellular transport and sorting of nerve growth factor and brain-derived neurotrophic factor. Together, these results suggest that α-syn might participate in mechanisms of vulnerability of selected neuronal populations in AD and that reducing α-syn might be a potential approach to protecting these populations from the toxic effects of Aβ., Significance Statement: Reducing endogenous α-synuclein (α-syn) in an APP transgenic mouse model of Alzheimer's disease (AD) prevented the degeneration of cholinergic neurons, ameliorated corresponding deficits, and recovered the levels of Rab3a and Rab5 proteins involved in intracellular transport and sorting of nerve growth factor and brain-derived neurotrophic factor. These results suggest that α-syn might participate in mechanisms of vulnerability of selected neuronal populations in AD and that reducing α-syn might be a potential approach to protecting these populations from the toxic effects of amyloid β., (Copyright © 2016 the authors 0270-6474/16/367971-14$15.00/0.)

Neurotoxicity of human immunodeficiency virus-1 (HIV) includes synaptic simplification and neuronal apoptosis. However, the mechanisms of HIV-associated neurotoxicity remain unclear, thus precluding an effective treatment of the neurological complications. The present study was undertaken to characterize novel mechanisms of HIV neurotoxicity that may explain how HIV subjects develop neuronal degeneration. Several neurodegenerative disorders are characterized by mitochondrial dysfunction; therefore, we hypothesized that HIV promotes mitochondrial damage. We first analyzed brains from HIV encephalitis (HIVE) by electron microscopy. Several sections of HIVE subjects contained enlarged and damaged mitochondria compared to brains from HIV subjects with no neurological complications. Similar pathologies were observed in mice overexpressing the HIV protein gp120, suggesting that this viral protein may be responsible for mitochondrial pathology found in HIVE. To gain more information about the cellular mechanisms of gp120 neurotoxicity, we exposed rat cortical neurons to gp120 and we determined cellular oxygen consumption rate, mitochondrial distribution, and trafficking. Our data show that gp120 evokes impairment in mitochondrial function and distribution. These data suggest that one of the mechanisms of HIV neurotoxicity includes altered mitochondrial dynamics in neurons.

A152T-variant human tau (hTau-A152T) increases risk for tauopathies, including Alzheimer's disease. Comparing mice with regulatable expression of hTau-A152T or wild-type hTau (hTau-WT), we find age-dependent neuronal loss, cognitive impairments, and spontaneous nonconvulsive epileptiform activity primarily in hTau-A152T mice. However, overexpression of either hTau species enhances neuronal responses to electrical stimulation of synaptic inputs and to an epileptogenic chemical. hTau-A152T mice have higher hTau protein/mRNA ratios in brain, suggesting that A152T increases production or decreases clearance of hTau protein. Despite their functional abnormalities, aging hTau-A152T mice show no evidence for accumulation of insoluble tau aggregates, suggesting that their dysfunctions are caused by soluble tau. In human amyloid precursor protein (hAPP) transgenic mice, co-expression of hTau-A152T enhances risk of early death and epileptic activity, suggesting copathogenic interactions between hTau-A152T and amyloid-β peptides or other hAPP metabolites. Thus, the A152T substitution may augment risk for neurodegenerative diseases by increasing hTau protein levels, promoting network hyperexcitability, and synergizing with the adverse effects of other pathogenic factors., (© 2016 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Lewy bodies (LBs) are intraneuronal inclusions consisting primarily of fibrillized human α-synuclein (hα-Syn) protein, which represent the major pathological hallmark of Parkinson's disease (PD). Although doubling hα-Syn expression provokes LB pathology in humans, hα-Syn overexpression does not trigger the formation of fibrillar LB-like inclusions in mice. We hypothesized that interactions between exogenous hα-Syn and endogenous mouse synuclein homologs could be attenuating hα-Syn fibrillization in mice, and therefore, we systematically assessed hα-Syn aggregation propensity in neurons derived from α-Syn-KO, β-Syn-KO, γ-Syn-KO, and triple-KO mice lacking expression of all three synuclein homologs. Herein, we show that hα-Syn forms hyperphosphorylated (at S129) and ubiquitin-positive LB-like inclusions in primary neurons of α-Syn-KO, β-Syn-KO, and triple-KO mice, as well as in transgenic α-Syn-KO mouse brains in vivo. Importantly, correlative light and electron microscopy, immunogold labeling, and thioflavin-S binding established their fibrillar ultrastructure, and fluorescence recovery after photobleaching/photoconversion experiments showed that these inclusions grow in size and incorporate soluble proteins. We further investigated whether the presence of homologous α-Syn species would interfere with the seeding and spreading of α-Syn pathology. Our results are in line with increasing evidence demonstrating that the spreading of α-Syn pathology is most prominent when the injected preformed fibrils and host-expressed α-Syn monomers are from the same species. These findings provide insights that will help advance the development of neuronal and in vivo models for understanding mechanisms underlying hα-Syn intraneuronal fibrillization and its contribution to PD pathogenesis, and for screening pharmacologic and genetic modulators of α-Syn fibrillization in neurons.

HIV-associated neurocognitive disorders (HAND) still occur in approximately 50% of HIV patients, and therapies to combat HAND progression are urgently needed. HIV proteins are released from infected cells and cause neuronal damage, possibly through mitochondrial abnormalities. Altered mitochondrial fission and fusion is implicated in several neurodegenerative disorders. Here, we hypothesized that mitochondrial fission/fusion may be dysregulated in neurons during HAND. We have identified decreased mitochondrial fission protein (dynamin 1-like; DNM1L) in frontal cortex tissues of HAND donors, along with enlarged and elongated mitochondria localized to the soma of damaged neurons. Similar pathology was observed in the brains of GFAP-gp120 tg mice. In vitro, recombinant gp120 decreased total and active DNM1L levels, reduced the level of Mitotracker staining, and increased extracellular acidification rate (ECAR) in primary neurons. DNM1L knockdown enhanced the effects of gp120 as measured by reduced Mitotracker signal in the treated cells. Interestingly, overexpression of DNM1L increased the level of Mitotracker staining in primary rat neurons and reduced neuroinflammation and neurodegeneration in the GFAP-gp120-tg mice. These data suggest that mitochondrial biogenesis dynamics are shifted towards mitochondrial fusion in brains of HAND patients and this may be due to gp120-induced reduction in DNM1L activity. Promoting mitochondrial fission during HIV infection of the CNS may restore mitochondrial biogenesis and prevent neurodegeneration., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene, which encodes a polyglutamine tract in the HTT protein. We found that peroxisome proliferator-activated receptor delta (PPAR-δ) interacts with HTT and that mutant HTT represses PPAR-δ-mediated transactivation. Increased PPAR-δ transactivation ameliorated mitochondrial dysfunction and improved cell survival of neurons from mouse models of HD. Expression of dominant-negative PPAR-δ in the central nervous system of mice was sufficient to induce motor dysfunction, neurodegeneration, mitochondrial abnormalities and transcriptional alterations that recapitulated HD-like phenotypes. Expression of dominant-negative PPAR-δ specifically in the striatum of medium spiny neurons in mice yielded HD-like motor phenotypes, accompanied by striatal neuron loss. In mouse models of HD, pharmacologic activation of PPAR-δ using the agonist KD3010 improved motor function, reduced neurodegeneration and increased survival. PPAR-δ activation also reduced HTT-induced neurotoxicity in vitro and in medium spiny-like neurons generated from stem cells derived from individuals with HD, indicating that PPAR-δ activation may be beneficial in HD and related disorders.

HIV-associated neurocognitive disorder (HAND) consists of motor and cognitive dysfunction in a relatively large percentage of patients with AIDS. Prior work has suggested that at least part of the neuronal and synaptic damage observed in HAND may occur due to excessive stimulation of NMDA-type glutamate receptors (NMDARs). Here, we compared pharmacological and genetic manipulation of NMDAR activity using an improved derivative of the NMDAR antagonist memantine, termed NitroMemantine, and the modulatory NMDAR subunit GluN3A in the HIV/gp120 transgenic (tg) mouse model of HAND. Interestingly, we found that while both NitroMemantine and GluN3A have been shown to inhibit NMDAR activity, NitroMemantine protected synapses in gp120-tg mice, but overexpression of GluN3A augmented the damage. Given recent findings in the field, one explanation for this apparently paradoxical result is the location of the NMDARs primarily affected, with NitroMemantine inhibiting predominantly extrasynaptic pathologically activated NMDARs, but GluN3A disrupting normal NMDAR-mediated neuroprotective activity via inhibition of synaptic NMDARs.

Cyanide is a life-threatening, bioterrorist agent, preventing cellular respiration by inhibiting cytochrome c oxidase, resulting in cardiopulmonary failure, hypoxic brain injury, and death within minutes. However, even after treatment with various antidotes to protect cytochrome oxidase, cyanide intoxication in humans can induce a delayed-onset neurological syndrome that includes symptoms of Parkinsonism. Additional mechanisms are thought to underlie cyanide-induced neuronal damage, including generation of reactive oxygen species. This may account for the fact that antioxidants prevent some aspects of cyanide-induced neuronal damage. Here, as a potential preemptive countermeasure against a bioterrorist attack with cyanide, we tested the CNS protective effect of carnosic acid (CA), a pro-electrophilic compound found in the herb rosemary. CA crosses the blood-brain barrier to up-regulate endogenous antioxidant enzymes via activation of the Nrf2 transcriptional pathway. We demonstrate that CA exerts neuroprotective effects on cyanide-induced brain damage in cultured rodent and human-induced pluripotent stem cell-derived neurons in vitro, and in vivo in various brain areas of a non-Swiss albino mouse model of cyanide poisoning that simulates damage observed in the human brain. Cyanide, a potential bioterrorist agent, can produce a chronic delayed-onset neurological syndrome that includes symptoms of Parkinsonism. Here, cyanide poisoning treated with the proelectrophillic compound carnosic acid, results in reduced neuronal cell death in both in vitro and in vivo models through activation of the Nrf2/ARE transcriptional pathway. Carnosic acid is therefore a potential treatment for the toxic central nervous system (CNS) effects of cyanide poisoning. ARE, antioxidant responsive element; Nrf2 (NFE2L2, Nuclear factor (erythroid-derived 2)-like 2)., (© 2015 International Society for Neurochemistry.)

Antiretroviral therapy has increased the life span of HIV+ individuals; however, HIV-associated neurocognitive disorder (HAND) occurrence is increasing in aging HIV patients. Previous studies suggest HIV infection alters autophagy function in the aging CNS and HIV-1 proteins affect autophagy in monocyte-derived cells. Despite these findings, the mechanisms leading to dysregulated autophagy in the CNS remain unclear. Here we sought to determine how HIV Tat dysregulates autophagy in neurons. Tat caused a dose-dependent decrease in autophagosome markers, microtubule-associated protein-1 light chain β II (LC3II), and sequestosome 1(SQSTM1), in a membrane-enriched fraction, suggesting Tat increases autophagic degradation. Bafilomycin A1 increased autophagosome number, LC3II, and SQSTM1 accumulation; Tat cotreatment diminished this effect. Tat had no effect when 3-methyladenine or knockdown of beclin 1 blocked early stages of autophagy. Tat increased numbers of LC3 puncta and resulted in the formation of abnormal autophagosomes in vitro. Likewise, in vivo studies in GFAP-Tat tg mice showed increased autophagosome accumulation in neurons, altered LC3II levels, and neurodegeneration. These effects were reversed by rapamycin treatment. Tat colocalized with autophagosome and lysosomal markers and enhanced the colocalization of autophagosome with lysosome markers. Furthermore, co-IP studies showed that Tat interacts with lysosomal-associated membrane protein 2A (LAMP2A) in vitro and in vivo, and LAMP2A overexpression reduces Tat-induced neurotoxicity. Hence, Tat protein may induce autophagosome and lysosome fusion through interaction with LAMP2A leading to abnormal neuronal autophagy function and dysregulated degradation of critical intracellular components. Therapies targeting Tat-mediated autophagy alterations may decrease neurodegeneration in aging patients with HAND., (Copyright © 2015 the authors 0270-6474/15/351921-18$15.00/0.)

Neurogranin is a calmodulin binding protein that has been implicated in learning and memory, long-term potentiation and synaptic plasticity. Neurons expressing neurogranin in the cortex degenerate in late stages of Parkinson's disease with widespread α-synuclein pathology. While analyzing neurogranin gene expression levels through rtPCR in brains of mouse models overexpressing human α-synuclein, we found levels were elevated 2.5 times when compared to nontransgenic animals. Immunohistochemistry in the cortex revealed colocalization between α-synuclein and neurogranin in mouse transgenics when compared to control mice. Coimmunoprecipitation studies in the superior temporal cortex in humans confirmed interaction between α-synuclein and neurogranin, and decreased interaction between α-synuclein and neurogranin was noticed in patients diagnosed with Parkinson's disease when compared to normal control brains. Additionally, phosphorylated neurogranin levels were also decreased in the human superior temporal cortex in patients diagnosed with Parkinson's disease and patients diagnosed with dementia with Lewy bodies. Here, we show for the first time that neurogranin binds to α-synuclein in the human cortex, and this interaction decreases in Parkinson's disease along with the phosphorylation of neurogranin, a molecular process thought to be involved in learning and memory., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background and Purpose: Anti-retrovirals have improved and extended the life expectancy of patients with HIV. However, as this population ages, the prevalence of cognitive changes is increasing. Aberrant activation of kinases, such as receptor tyrosine kinases (RTKs) and cyclin-dependent kinase 5 (CDK5), play a role in the mechanisms of HIV neurotoxicity. Inhibitors of CDK5, such as roscovitine, have neuroprotective effects; however, CNS penetration is low. Interestingly, tyrosine kinase inhibitors (TKIs) display some CDK inhibitory activity and ability to cross the blood-brain barrier., Experimental Approach: We screened a small group of known TKIs for a candidate with additional CDK5 inhibitory activity and tested the efficacy of the candidate in in vitro and in vivo models of HIV-gp120 neurotoxicity., Key Results: Among 12 different compounds, sunitinib inhibited CDK5 with an IC50 of 4.2 μM. In silico analysis revealed that, similarly to roscovitine, sunitinib fitted 6 of 10 features of the CDK5 pharmacophore. In a cell-based model, sunitinib reduced CDK5 phosphorylation (pCDK5), calpain-dependent p35/p25 conversion and protected neuronal cells from the toxic effects of gp120. In glial fibrillary acidic protein-gp120 transgenic (tg) mice, sunitinib reduced levels of pCDK5, p35/p25 and phosphorylated tau protein, along with amelioration of the neurodegenerative pathology., Conclusions and Implications: Compounds such as sunitinib with dual kinase inhibitory activity could ameliorate the cognitive impairment associated with chronic HIV infection of the CNS. Moreover, repositioning existing low MW compounds holds promise for the treatment of patients with neurodegenerative disorders., (© 2014 The British Pharmacological Society.)

Background: In dementia with Lewy bodies (DLB) abnormal interactions between α-synuclein (α-syn) and beta amyloid (Aβ) result in selective degeneration of neurons in the neocortex, limbic system and striatum. However, factors rendering these neurons selectively vulnerable have not been fully investigated. The metabotropic glutamate receptor 5 (mGluR5) has been shown to be up regulated in DLB and might play a role as a mediator of the neurotoxic effects of Aβ and α-syn in vulnerable neuronal populations. In this context, the main objective of the present study was to investigate the role of mGluR5 as a mediator of the neurotoxic effects of α-syn and Aβ in the hippocampus., Results: We generated double transgenic mice over-expressing amyloid precursor protein (APP) and α-syn under the mThy1 cassette and investigated the relationship between α-syn cleavage, Aβ, mGluR5 and neurodegeneration in the hippocampus. We found that compared to the single tg mice, the α-syn/APP tg mice displayed greater accumulation of α-syn and mGluR5 in the CA3 region of the hippocampus compared to the CA1 and other regions. This was accompanied by loss of CA3 (but not CA1) neurons in the single and α-syn/APP tg mice and greater loss of MAP 2 and synaptophysin in the CA3 in the α-syn/APP tg. mGluR5 gene transfer using a lentiviral vector into the hippocampus CA1 region resulted in greater α-syn accumulation and neurodegeneration in the single and α-syn/APP tg mice. In contrast, silencing mGluR5 with a lenti-shRNA protected neurons in the CA3 region of tg mice. In vitro, greater toxicity was observed in primary hippocampal neuronal cultures treated with Aβ oligomers and over-expressing α-syn; this effect was attenuated by down-regulating mGluR5 with an shRNA lentiviral vector. In α-syn-expressing neuronal cells lines, Aβ oligomers promoted increased intracellular calcium levels, calpain activation and α-syn cleavage resulting in caspase-3-dependent cell death. Treatment with pharmacological mGluR5 inhibitors such as 2-Methyl-6-(phenylethynyl)pyridine (MPEP) and 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP) attenuated the toxic effects of Aβ in α-syn-expressing neuronal cells., Conclusions: Together, these results support the possibility that vulnerability of hippocampal neurons to α-syn and Aβ might be mediated via mGluR5. Moreover, therapeutical interventions targeting mGluR5 might have a role in DLB.

In Parkinson's disease and dementia with Lewy bodies, α-synuclein aggregates to form oligomers and fibrils; however, the precise nature of the toxic α-synuclein species remains unclear. A number of synthetic α-synuclein mutations were recently created (E57K and E35K) that produce species of α-synuclein that preferentially form oligomers and increase α-synuclein-mediated toxicity. We have shown that acute lentiviral expression of α-synuclein E57K leads to the degeneration of dopaminergic neurons; however, the effects of chronic expression of oligomer-prone α-synuclein in synapses throughout the brain have not been investigated. Such a study could provide insight into the possible mechanism(s) through which accumulation of α-synuclein oligomers in the synapse leads to neurodegeneration. For this purpose, we compared the patterns of neurodegeneration and synaptic damage between a newly generated mThy-1 α-synuclein E57K transgenic mouse model that is prone to forming oligomers and the mThy-1 α-synuclein wild-type mouse model (Line 61), which accumulates various forms of α-synuclein. Three lines of α-synuclein E57K (Lines 9, 16 and 54) were generated and compared with the wild-type. The α-synuclein E57K Lines 9 and 16 were higher expressings of α-synuclein, similar to α-synuclein wild-type Line 61, and Line 54 was a low expressing of α-synuclein compared to Line 61. By immunoblot analysis, the higher-expressing α-synuclein E57K transgenic mice showed abundant oligomeric, but not fibrillar, α-synuclein whereas lower-expressing mice accumulated monomeric α-synuclein. Monomers, oligomers, and fibrils were present in α-synuclein wild-type Line 61. Immunohistochemical and ultrastructural analyses demonstrated that α-synuclein accumulated in the synapses but not in the neuronal cells bodies, which was different from the α-synuclein wild-type Line 61, which accumulates α-synuclein in the soma. Compared to non-transgenic and lower-expressing mice, the higher-expressing α-synuclein E57K mice displayed synaptic and dendritic loss, reduced levels of synapsin 1 and synaptic vesicles, and behavioural deficits. Similar alterations, but to a lesser extent, were seen in the α-synuclein wild-type mice. Moreover, although the oligomer-prone α-synuclein mice displayed neurodegeneration in the frontal cortex and hippocampus, the α-synuclein wild-type only displayed neuronal loss in the hippocampus. These results support the hypothesis that accumulating oligomeric α-synuclein may mediate early synaptic pathology in Parkinson's disease and dementia with Lewy bodies by disrupting synaptic vesicles. This oligomer-prone model might be useful for evaluating therapies directed at oligomer reduction.

Background: Mutations in the gene encoding parkin, a neuroprotective protein with dual functions as an E3 ubiquitin ligase and transcriptional repressor of p53, are linked to familial forms of Parkinson's disease (PD). We hypothesized that oxidative posttranslational modification of parkin by environmental toxins may contribute to sporadic PD., Results: We first demonstrated that S-nitrosylation of parkin decreased its activity as a repressor of p53 gene expression, leading to upregulation of p53. Chromatin immunoprecipitation as well as gel-shift assays showed that parkin bound to the p53 promoter, and this binding was inhibited by S-nitrosylation of parkin. Additionally, nitrosative stress induced apoptosis in cells expressing parkin, and this death was, at least in part, dependent upon p53. In primary mesencephalic cultures, pesticide-induced apoptosis was prevented by inhibition of nitric oxide synthase (NOS). In a mouse model of pesticide-induced PD, both S-nitrosylated (SNO-)parkin and p53 protein levels were increased, while administration of a NOS inhibitor mitigated neuronal death in these mice. Moreover, the levels of SNO-parkin and p53 were simultaneously elevated in postmortem human PD brain compared to controls., Conclusions: Taken together, our data indicate that S-nitrosylation of parkin, leading to p53-mediated neuronal cell death, contributes to the pathophysiology of sporadic PD.

Proteostasis is critical for maintaining cell function and proteome stability may play an important role in human embryonic stem cell (hESC) immortality. Notably, hESC populations exhibit a high assembly of active proteasomes, a key node of the proteostasis network. FOXO4, an insulin/IGF-1 responsive transcription factor, regulates proteasome activity in hESCs. We find that loss of FOXO4 reduces the potential of hESCs to differentiate into neural lineages. Therefore, FOXO4 crosses evolutionary boundaries and links hESC function to invertebrate longevity modulation.

Modifications to the gene encoding human α-synuclein have been linked to the development of Parkinson's disease. The highly conserved structure of α-synuclein suggests a functional interaction with membranes, and several lines of evidence point to a role in vesicle-related processes within nerve terminals. Using recombinant fusions of human α-synuclein, including new genetic tags developed for correlated light microscopy and electron microscopy (the tetracysteine-biarsenical labeling system or the new fluorescent protein for electron microscopy, MiniSOG), we determined the distribution of α-synuclein when overexpressed in primary neurons at supramolecular and cellular scales in three dimensions (3D). We observed specific association of α-synuclein with a large and otherwise poorly characterized membranous organelle system of the presynaptic terminal, as well as with smaller vesicular structures within these boutons. Furthermore, α-synuclein was localized to multiple elements of the protein degradation pathway, including multivesicular bodies in the axons and lysosomes within neuronal cell bodies. Examination of synapses in brains of transgenic mice overexpressing human α-synuclein revealed alterations of the presynaptic endomembrane systems similar to our findings in cell culture. Three-dimensional electron tomographic analysis of enlarged presynaptic terminals in several brain areas revealed that these terminals were filled with membrane-bounded organelles, including tubulovesicular structures similar to what we observed in vitro. We propose that α-synuclein overexpression is associated with hypertrophy of membrane systems of the presynaptic terminal previously shown to have a role in vesicle recycling. Our data support the conclusion that α-synuclein is involved in processes associated with the sorting, channeling, packaging, and transport of synaptic material destined for degradation.

Abnormal aggregation of α-synuclein and sustained microglial activation are important contributors to the pathogenic processes of Parkinson's disease. However, the relationship between disease-associated protein aggregation and microglia-mediated neuroinflammation remains unknown. Here, using a combination of in silico, in vitro and in vivo approaches, we show that extracellular α-synuclein released from neuronal cells is an endogenous agonist for Toll-like receptor 2 (TLR2), which activates inflammatory responses in microglia. The TLR2 ligand activity of α-synuclein is conformation-sensitive; only specific types of oligomer can interact with and activate TLR2. This paracrine interaction between neuron-released oligomeric α-synuclein and TLR2 in microglia suggests that both of these proteins are novel therapeutic targets for modification of neuroinflammation in Parkinson's disease and related neurological diseases.

α-Synuclein has been reported to be important in modulating brain plasticity and to be a key protein in neurodegenerative diseases, including Lewy body dementia (LBD). We investigated how α-synuclein levels modulate adult neurogenesis and the development of dendritic arborization and spines in the dentate gyrus, in which new neurons are constantly added. In the human hippocampus, levels of endogenous α-synuclein were increased in LBD, and the numbers of SOX2-positive cells were decreased. We investigated whether newly generated neurons were modulated by endogenous α-synuclein, and we found increased adult neurogenesis in α/β-synuclein knock-out mice. In contrast, overexpression of human wild-type α-synuclein (WTS) decreased the survival and dendritic development of newborn neurons. Endogenous α-synuclein expression levels increased the negative impact of WTS on dendrite development, suggesting a toxic effect of increasing amounts of α-synuclein. To attempt a rescue of the dendritic phenotype, we administered rolipram to activate the cAMP response element-binding protein pathway, which led to a partial rescue of neurite development. The current work provides novel insights into the role of α-synuclein in adult hippocampal neurogenesis.

Parkinson disease is characterized by the loss of dopaminergic neurons mainly in the substantia nigra. Accumulation of α-synuclein and cell loss has been also reported in many other brain regions including the hippocampus, where it might impair adult neurogenesis, contributing to nonmotor symptoms. However, the molecular mechanisms of these alterations are still unknown. In this report we show that α-synuclein-accumulating adult rat hippocampus neural progenitors present aberrant neuronal differentiation, with reduction of Notch1 expression and downstream signaling targets. We characterized a Notch1 proximal promoter that contains p53 canonical response elements. In vivo binding of p53 represses the transcription of Notch1 in neurons. Moreover, we demonstrated that α-synuclein directly binds to the DNA at Notch1 promoter vicinity and also interacts with p53 protein, facilitating or increasing Notch1 signaling repression, which interferes with maturation and survival of neural progenitors cells. This study provides a molecular basis for α-synuclein-mediated disruption of adult neurogenesis in Parkinson disease.

Sestrin-2 is involved in p53-dependent antioxidant defenses and in the maintenance of metabolic homeostasis. We hypothesize that sestrin-2 expression is altered in the brains of subjects diagnosed with human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) due to neuronal oxidative stress. We studied sestrin-2 immunoreactivity in 42 isocortex sections from HIV-1-infected subjects compared to 18 age-matched non-HIV controls and 19 advanced Alzheimer's disease (AD) cases. With HIV infection, the sestrin-2 immunoreactivity pattern shifted from neuropil predominance (N) to neuropil and neuronal-soma co-dominance (NS) and neuronal-soma predominance (S; P < 0.0001, Chi-square test for linear trend). Among HIV cases showing the NS or S pattern, HAND cases were preferentially associated with the S pattern (n = 10 of 20) compared to cognitively intact cases (n = 1 of 11; P = 0.047, Fisher's exact test). In AD brains, sestrin-2 immunoreactivity was mostly intense in the neuropil and co-localized with phospho-Tau immunoreactivity in a subset of neurofibrillary lesions. Phospho-Tau-immunoreactive neurofibrillary lesions were rare in HIV cases and their occurrence was not associated with HAND. Levels of isocortical 8-hydroxy-deoxyguanosine (marker of nucleic acid oxidation) immunoreactivity were not significantly altered in HAND cases compared to cognitively intact HIV cases. In conclusion, the sestrin-2 immunoreactivity redistribution to neuronal soma in HAND suggests unique involvement of sestrin-2 in the pathophysiology of HAND, which is different from the role of sestrin-2 in AD pathogenesis. Alternatively, the difference in sestrin-2 immunoreactivity distribution between HAND and AD may be related to different degrees of severity or stages of oxidative stress.

Abnormal accumulation of α-synuclein is centrally involved in the pathogenesis of many disorders with Parkinsonism and dementia. Previous in vitro studies suggest that α-synuclein dysregulates intracellular calcium. However, it is unclear whether these alterations occur in vivo. For this reason, we investigated calcium dynamics in transgenic mice expressing human WT α-synuclein using two-photon microscopy. We imaged spontaneous and stimulus-induced neuronal activity in the barrel cortex. Transgenic mice exhibited augmented, long-lasting calcium transients characterized by considerable deviation from the exponential decay. The most evident pathology was observed in response to a repetitive stimulation in which subsequent stimuli were presented before relaxation of calcium signal to the baseline. These alterations were detected in the absence of significant increase in neuronal spiking response compared with age-matched controls, supporting the possibility that α-synuclein promoted alterations in calcium dynamics via interference with intracellular buffering mechanisms. The characteristic shape of calcium decay and augmented response during repetitive stimulation can serve as in vivo imaging biomarkers in this model of neurodegeneration, to monitor progression of the disease and screen candidate treatment strategies.

Multiple system atrophy is a sporadic, progressive, neurodegenerative disease characterized by an oligodendroglial accumulation of alpha-synuclein (α-syn). The mechanisms underlying the oligodendroglial accumulation of α-syn in the brains of patients with multiple system atrophy have attracted a great deal of interest, given the primarily neuronal role reported for this protein. We examined the interactions between neuronal and oligodendroglial α-syn in the progeny of crosses between parental transgenic (tg) mouse lines that express α-syn either under the oligodendroglial-specific myelin-basic protein promoter (MBP1-hα-syn tg) or under the neuronal platelet-derived growth factor promoter (PDGF-hα-syn tg). Our results demonstrate that progeny from the cross [hα-syn double (dbl) tg mice] displayed a robust redistribution of α-syn accumulation, with a relocalization from a neuronal or a mixed neuronal/oligodendroglial α-syn expression to a more oligodendroglial pattern in both the neocortex and the basal ganglia that closely resembled the parental MBP-hα-syn tg line. The hα-syn dbl tg mice also displayed motor deficits, concomitant with reduced levels of tyrosine hydroxylase and augmented neuropathological alterations in the basal ganglia. These results suggest that the central nervous system milieu in the hα-syn dbl tg mice favors an oligodendroglial accumulation of α-syn. This model represents an important tool to examine the interactions between neuronal and oligodendrocytic α-syn in diseases such as multiple system atrophy.

Neonatal ventral hippocampal lesion (nVHL) in rats has been widely used as a neurodevelopmental model to mimic schizophrenia-like behaviors. Recently, we reported that nVHLs result in dendritic retraction and spine loss in prefrontal cortex (PFC) pyramidal neurons and medium spiny neurons of the nucleus accumbens (NAcc). Cerebrolysin (Cbl), a neurotrophic peptide mixture, has been reported to ameliorate the synaptic and dendritic pathology in models of aging and neurodevelopmental disorder such as Rett syndrome. This study sought to determine whether Cbl was capable of reducing behavioral and neuronal alterations in nVHL rats. The behavioral analysis included locomotor activity induced by novel environment and amphetamine, social interaction, and sensoriomotor gating. The morphological evaluation included dendritic analysis by using the Golgi-Cox procedure and stereology to quantify the total cell number in PFC and NAcc. Behavioral data show a reduction in the hyperresponsiveness to novel environment- and amphetamine-induced locomotion, with an increase in the total time spent in social interactions and in prepulse inhibition in Cbl-treated nVHL rats. In addition, neuropathological analysis of the limbic regions also showed amelioration of dendritic retraction and spine loss in Cbl-treated nVHL rats. Cbl treatment also ameliorated dendritic pathology and neuronal loss in the PFC and NAcc in nVHL rats. This study demonstrates that Cbl promotes behavioral improvements and recovery of dendritic neuronal damage in postpubertal nVHL rats and suggests that Cbl may have neurotrophic effects in this neurodevelopmental model of schizophrenia. These findings support the possibility that Cbl has beneficial effects in the management of schizophrenia symptoms., (Copyright © 2011 Wiley Periodicals, Inc.)

Background: Alpha synuclein (SNCA) has been linked to neurodegenerative diseases (synucleinopathies) that include Parkinson's disease (PD). Although the primary neurodegeneration in PD involves nigrostriatal dopaminergic neurons, more extensive yet regionally selective neurodegeneration is observed in other synucleinopathies. Furthermore, SNCA is ubiquitously expressed in neurons and numerous neuronal systems are dysfunctional in PD. Therefore it is of interest to understand how overexpression of SNCA affects neuronal function in regions not directly targeted for neurodegeneration in PD., Results: The present study investigated the consequences of SNCA overexpression on cellular processes and functions in the striatum of mice overexpressing wild-type, human SNCA under the Thy1 promoter (Thy1-aSyn mice) by transcriptome analysis. The analysis revealed alterations in multiple biological processes in the striatum of Thy1-aSyn mice, including synaptic plasticity, signaling, transcription, apoptosis, and neurogenesis., Conclusion: The results support a key role for SNCA in synaptic function and revealed an apoptotic signature in Thy1-aSyn mice, which together with specific alterations of neuroprotective genes suggest the activation of adaptive compensatory mechanisms that may protect striatal neurons in conditions of neuronal overexpression of SNCA.

Polyglutamine (polyQ) stretches exceeding a threshold length confer a toxic function to proteins that contain them and cause at least nine neurological disorders. The basis for this toxicity threshold is unclear. Although polyQ expansions render proteins prone to aggregate into inclusion bodies, this may be a neuronal coping response to more toxic forms of polyQ. The exact structure of these more toxic forms is unknown. Here we show that the monoclonal antibody 3B5H10 recognizes a species of polyQ protein in situ that strongly predicts neuronal death. The epitope selectively appears among some of the many low-molecular-weight conformational states assumed by expanded polyQ and disappears in higher-molecular-weight aggregated forms, such as inclusion bodies. These results suggest that protein monomers and possibly small oligomers containing expanded polyQ stretches can adopt a conformation that is recognized by 3B5H10 and is toxic or closely related to a toxic species.

Proton magnetic resonance spectroscopy has emerged as one of the most informative neuroimaging modalities for studying the effect of HIV infection in the brain, providing surrogate markers by which to assess disease progression and monitor treatment. Reductions in the level of N-Acetylaspartate and N-Acetylaspartate/creatine are established markers of neuronal injury or loss. However, the biochemical basis of altered creatine levels in neuroAIDS is not well understood. This study used a rapid progression macaque model of neuroAIDS to elucidate the changes in creatine. As the disease progressed, proton magnetic resonance spectroscopy revealed a decrease in N-Acetylaspartate, indicative of neuronal injury, and an increase in creatine yet to be elucidated. Subsequently, immunohistochemistry and stereology measures of decreased synaptophysin, microtubule-associated protein 2, and neuronal density confirmed neuronal injury. Furthermore, increases in ionized calcium binding adaptor molecule 1 and glial fibrillary acidic protein indicated microglial and astroglial activation, respectively. Given these data, elevated creatine may reflect enhanced high-energy phosphate turnover in highly metabolizing activated astrocytes and microglia., (Copyright © 2011 Wiley-Liss, Inc.)

Transthyretin (TTR), a systemic amyloid precursor in the human TTR amyloidoses, interacts with β-amyloid (Aβ) in vitro, inhibits Aβ fibril formation, and suppresses the Alzheimer's disease (AD) phenotype in APP23 mice bearing a human APP gene containing the Swedish autosomal dominant AD mutation. In the present study, we show that TTR is a neuronal product upregulated in AD. Immunohistochemical analysis reveals that, in contrast to brains from non-demented age-matched individuals and control mice, the majority of hippocampal neurons from human AD and all those from the APP23 mouse brains contain TTR. Quantitative PCR for TTR mRNA and Western blot analysis show that primary neurons from APP23 mice transcribe TTR mRNA, and the cells synthesize and secrete TTR protein. TTR mRNA abundance is greatly increased in cultured cortical and hippocampal embryonic neurons and cortical lysates from adult APP23 mice. Antibodies specific for TTR and Aβ pulled down TTR/Aβ complexes from cerebral cortical extracts of APP23 mice and some human AD patients but not from control brains. In complementary tissue culture experiments, recombinant human TTR suppressed the cytotoxicity of soluble Aβ aggregates added to mouse neurons and differentiated human SH-SY5Y neuroblastoma cells. The findings that production of Aβ, its precursor, or its related peptides induces neuronal TTR transcription and synthesis and the presence of Aβ/TTR complexes in vivo suggest that increased TTR production coupled with interaction between TTR and Aβ and/or its related peptides may play a role in natural resistance to human AD.

The protein α-synuclein has a central role in Parkinson disease, but the mechanism by which it contributes to neural degeneration remains unknown. We now show that the expression of α-synuclein in mammalian cells, including neurons in vitro and in vivo, causes the fragmentation of mitochondria. The effect is specific for synuclein, with more fragmentation by α- than β- or γ-isoforms, and it is not accompanied by changes in the morphology of other organelles or in mitochondrial membrane potential. However, mitochondrial fragmentation is eventually followed by a decline in respiration and neuronal death. The fragmentation does not require the mitochondrial fission protein Drp1 and involves a direct interaction of synuclein with mitochondrial membranes. In vitro, synuclein fragments artificial membranes containing the mitochondrial lipid cardiolipin, and this effect is specific for the small oligomeric forms of synuclein. α-Synuclein thus exerts a primary and direct effect on the morphology of an organelle long implicated in the pathogenesis of Parkinson disease.

α-Synuclein (α-Syn) is a key protein that accumulates as hyperphosphorylated aggregates in pathologic hallmark features of Parkinson's disease (PD) and other neurodegenerative disorders. Phosphorylation of this protein at serine 129 is believed to promote its aggregation and neurotoxicity, suggesting that this post-translational modification could be a therapeutic target. Here, we demonstrate that phosphoprotein phosphatase 2A (PP2A) dephosphorylates α-Syn at serine 129 and that this activity is greatly enhanced by carboxyl methylation of the catalytic C subunit of PP2A. α-Syn-transgenic mice raised on a diet supplemented with eicosanoyl-5-hydroxytryptamide, an agent that enhances PP2A methylation, dramatically reduced both α-Syn phosphorylation at Serine 129 and α-Syn aggregation in the brain. These biochemical changes were associated with enhanced neuronal activity, increased dendritic arborizations, and reduced astroglial and microglial activation, as well as improved motor performance. These findings support the notion that serine 129 phosphorylation of α-Syn is of pathogenetic significance and that promoting PP2A activity is a viable disease-modifying therapeutic strategy for α-synucleinopathies such as PD.

Recent studies show that in Alzheimer's disease (AD), alterations in neurogenesis contribute to the neurodegenerative process. Neurodegeneration in AD has been associated with aberrant signaling through the cyclin-dependent kinase-5 (CDK5) pathway via its activators p35/p25; however, the role of CDK5 in the mechanisms of defective adult neurogenesis in AD is unknown. First, to study AD-like abnormal activation of CDK5 signaling in an in vitro model of neurogenesis, neuronal progenitor cells (NPCs) were infected with a viral vector expressing p35, and exposed to amyloid-β protein (Aβ(1-42)). These conditions resulted in impaired maturation and neurite outgrowth in vitro, and these effects were reversed by pharmacological or genetic inhibition of CDK5. Similarly, neurogenesis was impaired in a transgenic mouse model of AD that expresses high levels of amyloid precursor protein (APP), and this effect was reversed in transgenic mice crossed with a CDK5 heterozygous-deficient mouse line. A similar rescue effect was observed in APP transgenic mice treated with Roscovitine, a pharmacological inhibitor of CDK5. Taken together, these data suggest that the CDK5 signaling pathway has a critical role in maintaining the integrity of NPCs and neuronal maturation in the adult hippocampus. Moreover, potential therapeutic approaches could focus on modulating the aberrant activity of CDK5 to target the neurogenic and neurodegenerative alterations in AD.

The entorhinal cortex (EC) is one of the earliest affected, most vulnerable brain regions in Alzheimer's disease (AD), which is associated with amyloid-β (Aβ) accumulation in many brain areas. Selective overexpression of mutant amyloid precursor protein (APP) predominantly in layer II/III neurons of the EC caused cognitive and behavioral abnormalities characteristic of mouse models with widespread neuronal APP overexpression, including hyperactivity, disinhibition, and spatial learning and memory deficits. APP/Aβ overexpression in the EC elicited abnormalities in synaptic functions and activity-related molecules in the dentate gyrus and CA1 and epileptiform activity in parietal cortex. Soluble Aβ was observed in the dentate gyrus, and Aβ deposits in the hippocampus were localized to perforant pathway terminal fields. Thus, APP/Aβ expression in EC neurons causes transsynaptic deficits that could initiate the cortical-hippocampal network dysfunction in mouse models and human patients with AD., (Copyright © 2010 Elsevier Inc. All rights reserved.)

In Alzheimer's disease brains, morphological changes in the dendrites of pyramidal neurons of the prefrontal cortex (PFC) and hippocampus have been observed. These changes are particularly reflected in the decrement of both the dendritic tree and spine number. Donepezil is a potent and selective acetylcholinesterase inhibitor used in the treatment of Alzheimer's disease. We have studied the effect of oral administration of this drug on the morphology of neuronal cells from the brain of aged rats. We examined dendrites of pyramidal neurons of the PFC, dorsal or ventral hippocampus (VH), and medium spiny neurons of the nucleus accumbens (NAcc). Donepezil (1 mg/kg, vo) was administrated every day for 60 days to rats aged 10 and 18 months. Dendritic morphology was studied by the Golgi-Cox stain procedure followed by Sholl analysis at 12 and 20 months ages, respectively. In all Donepezil-treated rats, a significant increment of the dendritic spines number in pyramidal neurons of the PFC and dorsal hippocampus was observed. However, pyramidal neurons of the VH and medium spiny cells of the NAcc only showed an increase in the number of their spines in 12-month-old rats. Our results suggest that Donepezil prevents the alterations of the neuronal dendrite morphology caused by aging., ((c) 2010 Wiley-Liss, Inc.)

Objective: Prolonged human immunodeficiency virus-1 (HIV-1) infection leads to neurological debilitation, including motor dysfunction and frank dementia. Although pharmacological control of HIV infection is now possible, HIV-associated neurocognitive disorders (HAND) remain intractable. Here, we report that chronic treatment with erythropoietin (EPO) and insulin-like growth factor-I (IGF-I) protects against HIV/gp120-mediated neuronal damage in culture and in vivo., Methods: Initially, we tested the neuroprotective effects of various concentrations of EPO, IGF-I, or EPO+IGF-I from gp120-induced damage in vitro. To assess the chronic effects of EPO+IGF-I administration in vivo, we treated HIV/gp120-transgenic or wild-type mice transnasally once a week for 4 months and subsequently conducted immunohistochemical analyses., Results: Low concentrations of EPO+IGF-I provided neuroprotection from gp120 in vitro in a synergistic fashion. In vivo, EPO+IGF-I treatment prevented gp120-mediated neuronal loss, but did not alter microgliosis or astrocytosis. Strikingly, in the brains of both humans with HAND and gp120-transgenic mice, we found evidence for hyperphosphorylated tau protein (paired helical filament-I tau), which has been associated with neuronal damage and loss. In the mouse brain following transnasal treatment with EPO+IGF-I, in addition to neuroprotection we observed increased phosphorylation/activation of Akt (protein kinase B) and increased phosphorylation/inhibition of glycogen synthase kinase (GSK)-3beta, dramatically decreasing downstream hyperphosphorylation of tau. These results indicate that the peptides affected their cognate signaling pathways within the brain parenchyma., Interpretation: Our findings suggest that chronic combination therapy with EPO+IGF-I provides neuroprotection in a mouse model of HAND, in part, through cooperative activation of phosphatidylinositol 3-kinase/Akt/GSK-3beta signaling. This combination peptide therapy should therefore be tested in humans with HAND.

An expanded polyglutamine (polyQ) stretch in the protein huntingtin (htt) induces self-aggregation into inclusion bodies (IBs) and causes Huntington's disease (HD). Defining precise relationships between early observable variables and neuronal death at the molecular and cellular levels should improve our understanding of HD pathogenesis. Here, we used an automated microscope that tracks thousands of neurons individually over their entire lifetime to quantify interconnected relationships between early variables, such as htt levels, polyQ length, and IB formation, and neuronal death in a primary striatal model of HD. The resulting model revealed that mutant htt increases the risk of death by tonically interfering with homeostatic coping mechanisms rather than producing accumulated damage to the neuron, htt toxicity is saturable, the rate-limiting steps for inclusion body formation and death can be traced to different conformational changes in monomeric htt, and IB formation reduces the impact of the starting levels of htt of a neuron on its risk of death. Finally, the model that emerges from our quantitative measurements places critical limits on the potential mechanisms by which mutant htt might induce neurodegeneration, which should help direct future research.

The lipid transport protein apolipoprotein E (apoE) is abundantly expressed in the brain. Its main isoforms in humans are apoE2, apoE3, and apoE4. ApoE4 is the major known genetic risk factor for Alzheimer's disease and also contributes to the pathogenesis of various other neurological conditions. In the central nervous system, apoE is synthesized by glial cells and neurons, but it is unclear whether the cellular source affects its biological activities. To address this issue, we induced excitotoxic injury by systemic kainic acid injection in transgenic Apoe knockout mice expressing human apoE isoforms in astrocytes or neurons. Regardless of its cellular source, apoE3 expression protected neuronal synapses and dendrites against the excitotoxicity seen in apoE-deficient mice. Astrocyte-derived apoE4, which has previously been shown to have detrimental effects in vitro, was as excitoprotective as apoE3 in vivo. In contrast, neuronal expression of apoE4 was not protective and resulted in loss of cortical neurons after excitotoxic challenge, indicating that neuronal apoE4 promotes excitotoxic cell death. Thus, an imbalance between astrocytic (excitoprotective) and neuronal (neurotoxic) apoE4 expression may increase susceptibility to diverse neurological diseases involving excitotoxic mechanisms.

Several neurodegenerative diseases are typified by intraneuronal alpha-synuclein deposits, synaptic dysfunction, and dementia. While even modest alpha-synuclein elevations can be pathologic, the precise cascade of events induced by excessive alpha-synuclein and eventually culminating in synaptotoxicity is unclear. To elucidate this, we developed a quantitative model system to evaluate evolving alpha-synuclein-induced pathologic events with high spatial and temporal resolution, using cultured neurons from brains of transgenic mice overexpressing fluorescent-human-alpha-synuclein. Transgenic alpha-synuclein was pathologically altered over time and overexpressing neurons showed striking neurotransmitter release deficits and enlarged synaptic vesicles; a phenotype reminiscent of previous animal models lacking critical presynaptic proteins. Indeed, several endogenous presynaptic proteins involved in exocytosis and endocytosis were undetectable in a subset of transgenic boutons ("vacant synapses") with diminished levels in the remainder, suggesting that such diminutions were triggering the overall synaptic pathology. Similar synaptic protein alterations were also retrospectively seen in human pathologic brains, highlighting potential relevance to human disease. Collectively the data suggest a previously unknown cascade of events where pathologic alpha-synuclein leads to a loss of a number of critical presynaptic proteins, thereby inducing functional synaptic deficits.

alpha-Synuclein (alpha-Syn) is a presynaptic protein implicated in Parkinson's disease (PD). Mice overexpressing human wildtype (WT) alpha-Syn under the Thy1 promoter show high levels of alpha-Syn in cortical and subcortical regions, exhibit progressive sensorimotor anomalies, as well as non-motor abnormalities and are considered models of pre-manifest PD as there is little evidence of early loss of dopaminergic (DA) neurons. We used whole-cell patch clamp recordings from visually identified striatal medium-sized spiny neurons (MSSNs) in slices from alpha-Syn and WT littermate control mice at 35, 90 and 300 days of age to examine corticostriatal synaptic function. MSSNs displayed significant decreases in the frequency of spontaneous excitatory postsynaptic currents (EPSCs) in alpha-Syn mice at all ages. This difference persisted in the presence of tetrodotoxin, indicating it was independent of action potentials. Stimulation thresholds for evoking EPSCs were significantly higher and responses were smaller in alpha-Syn mice. These data suggest a decrease in neurotransmitter release at the corticostriatal synapse. At 90 days the frequency of spontaneous GABA(A) receptor-mediated synaptic currents was decreased in MSSNs but increased in cortical pyramidal neurons. These observations indicate that high levels of expression of alpha-Syn alter corticostriatal synaptic function early and they provide evidence for early synaptic dysfunction in a pre-manifest model of PD. Of importance, these changes are opposite to those found in DA-depletion models, suggesting that before degeneration of DA neurons in the substantia nigra synaptic adaptations occur at the corticostriatal synapse that may initiate subtle preclinical manifestations.

Multiple systems atrophy (MSA) is a neurodegenerative disorder characterized by oligodendrocytic accumulations of alpha-synuclein (alphasyn). Oxidative stress is a key mechanism proposed to underlie MSA pathology. To address the role of alphasyn modifications, over and above general oxidative modifications, this study examined the effects of 3-nitropropionic acid (3NP) administration, a technique used to model MSA, in knock-out mice lacking alphasyn (alphasynKO). Although susceptible to 3NP-induced oxidative stress, alphasynKO mice display reduced neuronal loss and dendritic pathology. The alphasynKO mice are resistant to 3NP-induced motor deficits and display attenuated loss of tyrosine hydroxylase and dopamine transporter striatal immunoreactivity. The results suggest that deficits in MSA are not due to general oxidative protein modification but in addition may be related to specific alphasyn modifications.

Abnormal neuronal aggregation of alpha-synuclein is implicated in the development of many neurological disorders, including Parkinson disease and dementia with Lewy bodies. Glial cells also show extensive alpha-synuclein pathology and may contribute to disease progression. However, the mechanism that produces the glial alpha-synuclein pathology and the interaction between neurons and glia in the disease-inflicted microenvironment remain unknown. Here, we show that alpha-synuclein proteins released from neuronal cells are taken up by astrocytes through endocytosis and form inclusion bodies. The glial accumulation of alpha-synuclein through the transmission of the neuronal protein was also demonstrated in a transgenic mouse model expressing human alpha-synuclein. Furthermore, astrocytes that were exposed to neuronal alpha-synuclein underwent changes in the gene expression profile reflecting an inflammatory response. Induction of pro-inflammatory cytokines and chemokines correlated with the extent of glial accumulation of alpha-synuclein. Together, these results suggest that astroglial alpha-synuclein pathology is produced by direct transmission of neuronal alpha-synuclein aggregates, causing inflammatory responses. This transmission step is thus an important mediator of pathogenic glial responses and could qualify as a new therapeutic target.