Michael K. Lee, PhD - US grants

Parkinson's disease (PD), a late-life movement disorder affecting >500,000 individuals, it is associated with abnormalities of dopaminergic neurons in the substantia nigra, pars compacta (SNpc) and loss of dopaminergic terminals in striatum. Although the causes of the majority of cases of PD are not known, genetic studies of familial PD (<10% of total cases of PD) have recently identified a missense mutation (A53T) in alpha-Synuclein (alpha-Syn). Moreover, alpha-Syn accumulates in Lewy bodies (LB), cytoplasmic inclusions characteristic of familial and sporadic PD. These findings indicate that alpha-Syn is an important component in the pathogenesis of disease. Although the functions of alpha-Syn are not known, it is abundant in neurons and is particularly enriched in the presynaptic terminals. In humans and rodents, beta Syn and gamma-Syn, members of the Syn family, are also expressed in nervous tissues. To gain a better understanding of the roles of different Syn isotypes in normal neuronal functions and the pathogenesis of PD, we will examine the expression patterns and cell biology of different Syn in human and rodent tissues, and in transgenic (Tg) mice expressing wild-type or A53T alpha- Syn, and in transfected cells in vitro. Specifically, we propose: to determine the levels of expression and localization of Syn isotypes in specific brain regions and neural systems in rodents and humans during development and aging; to generate Tg mice expressing high levels of wild- type or A53T human alpha-Syn and to analyze the character/evolution of abnormalities in mice that over-express the mutant; to study the expression, metabolism, and subcellular localization of Syn isotype in lines of cultured cells and in primary neuronal cultures from Tg mice; and to define the axonal transport and metabolism of endogenous and transgene- encoded Syn in vivo. The results of these studies will provide information critical to understanding the roles of Syn in normal brain and the behavior of A53T alpha-Syn in vitro and in vivo model systems, particularly in Tg mice. Information about the mechanisms of disease will then be translated into the design of new therapies that can be tested in model systems.

DESCRIPTION (provided by applicant): Ubiquitination is a post-translational modification of proteins that regulate a host of important cellular processes including degradation of cellular proteins, expression of genes, and protein/membrane trafficking. Ubiquitination of proteins and the specificity of ubiquitination are mediated by three classes of ubiquitin ligases (E1, E2, and E3). In addition to the ligases, a variety of deubiquitinating enzymes (DUBs) may regulate ubiquitination in cells. DUBs include ubiquitin carboxy-terminal hydrolase L1 (Uch-L1), a protein selectively expressed in neurons and in sertoli cells of testis. Defects in Ubiquitination/proteasomal mechanisms are implicated in the pathogenesis of many neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. In particular, pathogenic relationship between defects in ubiquitin/proteasomal pathway and degeneration of dopaminergic neurons are indicated by the fact that PD-associated mutations are identified in Parkin, an E3-ubiquitin ligase, and Uch-L1. In gad mutant mice, deletion of exon 7 and 8 of Uch-L1 gene leads to degeneration of neurons in the DRG and in the gracile nucleus and early lethality. However, because Uch-L1 is expressed at high levels in many neuronal populations, Uch-L1 function may be important in other neurons. Because of early lethality and general underlying movement defect in gad mice, the functional importance of Uch-L1 in a various neuronal population and as a function of aging can not be examined effectively. We propose generate a mice where the expression of Uch-L1 can be temporally and spatially regulated. Specifically, we will generate UchL1-floxed mice to conditionally silence Uch-L1 expression by mating to appropriate Cre expressing mice. As an initial test of our hypothesis, we will determine whether Uch-L1 activity is important for normal functioning and aging of the dopaminergic and noradrenergic neurons by mating. The IoxP-targeted Uch1 mice will be mated to pTH-Cre transgenic mice to silence Uch-L1 expression only in the dopaminergic and noradrenergic neurons.

Genetic and biochemical abnormalities of alpha-synuclein are directly implicated in the pathogenesis of Parkinson's disease (PD) and other alpha-synucleinopathies. Transgenic (Tg) A53T mutant alpha-synuclein mice develop adult-onset disease with a progressive motoric dysfunction leading to death. The affected mice exhibit many of the feaures of human alpha-synucleinopathies including neuronal accumulations of alpha-synuclein and ubiquitin in many neuronal populations and increased insolubility and biochemical alterations of alpha-synuclein. We propose the following aims to more fully define mechanisms of alpha-synuclein dependent in vivo degeneration of neuronal populations. 1. To characterize the temporal and spatial changes in the neurodegeneration associated cellular processes in Hu alpha-synuclein Tg mice. The mechanisms leading to neuronal dysfunction and death in alpha-synucleinopathies are not known. By detailed pathological analysis of alpha-synuclein Tg mice, we will determine potential cellular processes that are involved in alpha-synucleinopathy associated neurondegeneration in vivo. 2. To characterize human alpha-synuclein metabolism, truncation, phosphorylation as a function of neuronal cell types, aging, and alpha-synucleinopathy in mice. Changes in cellular metabolism and posttranslational modifications may by a significant factor in alpha-synucleinopathies. We will examine whether cell types, aging and disease states are associated with alpha-synuclein metabolism that promote alpha-synucleinopathies. 3. To characterize Hu alpha-synuclein metabolism, truncation, and aggregation as a function of neuronal cell type, aging, and disease in human brain. We will examine whether regional variations in alpha-synuclein metabolism/posttranslational modifications are associated with increased in human alpha-synucleinopathies. We will also determine whether alpha-synucleinopathies in humans are associated with alterations in metabolism, truncations, and aggregation of alpha-synuclein. 4. To determine whether alpha-synuclein metabolism and truncation is affected by Ser-129 phosphorylation, Synphilin, Parkin, and DJ-1. In collaboration with Projects 1, 2, and 4, this aim will examine whether other PD related processes and genes directly affect alpha-synuclein metabolism and truncation. 5. To determine whether the C-terminal truncations of Hu alpha-synuclein increases toxicity and aggregation of Hu alpha-synuclein in cultured cells and in transgenic mice. In conjunction with Drs. Chris Ross (Project 2) and Valina Dawson (Project 4), we will establish that C-terminally truncated alpha-synucleins shows enhanced toxicity and aggregation properties in cultured cells (SH-SY5Y, PC-12 and primary mesencephalic neurons). To establish the pathogenic importance in vivo, we will analyze Tg mice expressing truncated human alpha-synuclein (generated in Transgenic Core).

[unreadable] DESCRIPTION (provided by applicant): Genetic and biochemical abnormalities of a-synuclein (a-Syn) are directly implicated in the pathogenesis of familial and sporadic forms of Parkinson's disease (PD). We have show that transgenic (Tg) mice expressing the A53T mutant oc-Syn, but not wild type (WT) or A30P, develop adult-onset disease with a progressive motor dysfunction, shortened life-span, and pathological features of human a-synucleinopathies. The studies of these mice and the cell lines expressing a-Syn have allowed us to identify potentially pathogenic biochemical alterations of a-Syn. Specifically, the disease in Tg mice is associated with the increase levels of detergent insoluble a-Syn aggregates and the accumulation of potentially pathogenic soluble oligomers. Our studies show that a-Syn is normally proteolytically truncated at the Carboxy-terminal region to generate a-Syn(AC)s. Correlative studies in human tissues, cell lines, and in vitro aggregation studies support the view that a-Syn(AC)s promotes the pathological aggregation of a-Syn. Our results also show that differential stabilization of a-Syn polypeptides may modulate vulnerability to a-synucleinopathies. Thus, factors that affect a-Syn metabolism modulate the vulnerability of selected neuronal populations to a-synucleinopathies in humans and in mice. In this proposal, we will explore the relationships between a-Syn metabolism/truncation and aggregation/oligomerization of a-Syn. To better define the relationships between of a-Syn metabolism, a-Syn aggregation and a-synucleinopathies, we propose following Aims. 1) Characterize proteolytic truncations of a-Syn and a-synucleinopathy-associated alterations of a-Syn in human and mouse. 2) Characterize the aggregation and oligomerization properties of truncated a-Syn variants using in vitro and in cell culture systems. 3) Characterize the primary sequence determinants and the cell biology of a-Syn metabolism/truncations. 4) Determine whether the known proteolytic systems are responsible for a-Syn metabolism/truncation in neuronal cells. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): While the causes of Parkinson's disease is not known, genetic and biochemical abnormalities of a-synuclein (a-Syn) are directly implicated in the pathogenesis PD and other a-synucleinopathies. We have shown that transgenic (Tg) mice expressing the A53T mutant human a-Syn using the mouse prion protein promoter (mPrP), but not wild type (WT) or A30P, develop adult-onset disease with a progressive motoric dysfunction leading to death. The affected mice exhibit many features of human a-synucleinopathies, including fibrillar aggregation of a-Syn and neurodegeneration. While the pathogenic mechanisms of a-synucleinpathy is current not settled, number of studies indicate that modulation of cellular protein chaperones can alter toxicity associated with a-Syn expression/aggregation. In particular, increased levels of heat shock protein can protect neurons for a-Syn-dependent degeneration in the Drosophila model a-synucleinopathy. Thus, pharmacological induction of cellular chaperone expression could be therapeutic benefit for a-synucleinopathy. This rationale is supported by the fact that the pharmacological induction of HSP delays disease progression in SOD1 transgenic mouse model of ALS. Recently, Celasterol, a natural product derived from the Celastraceae family of plants, has been shown to be a potent activator or HSF-1 and HSP expression. We will test whether Celastrol can modulate a-synucleinopathy in the Transgenic (Tg) mice expressing the A53T mutant Hua-Syn and Dopaminergic degeneration in a chronic MPTP model of PD. The study will provide valuable mechanistic insights about the pathogenesis of a-synucleinopaty in vivo. More important, we hope to provide a strong rational for further screening and testing of other compounds that can induce HSP expression as potential therapeutic agents for treating a-synucleinopathy. Parkinson's Disease and related alpha-synucleinopathies are fatal neurodegenerative diseases affecting 500,000-1,000,000 individuals annually in US. Currently, there are no treatment to slow or halt the progression of these diseases. In the last several years, studies have implicated genetic and biochemical abnormalities of alpha-synuclein in the pathogenesis of PD. In vitro studies and studies in a fly model of alpha-synuclein dependent neurodegeneration suggest that increased expression of heat shock regulated chaperones could provide neuroprotection from the neurotoxic effect of alpha-synuclein abnormalities. We will determine whether increase in heat shock proteins, via administration of novel compound celastrol, can prevent neurodegeneration in mammalian animal models that are directly relevant to PD. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a common late onset, progressive neurodegenerative disease characterized by degeneration of subcortical neuronal populations, including dopaminergic neurons of substantia nigra, pars compacta (SNpc), and presence of the cytoplasmic inclusions composed of alpha-synuclein. While the causes of PD are not known, genetic and biochemical abnormalities of alpha-synuclein are directly implicated in the pathogenesis of PD and other alpha-synucleinopathies. Causal link between alpha-synuclein abnormalities and neurodegeneration is shown by various genetic models where alpha-synuclein abnormalities lead to adult- onset neurological disease with neurodegeneration. Some models exhibit many of the features of human alpha-synucleinopathies, including aberrant aggregation of alpha-synuclein and neurodegeneration in subcortical regions. Our studies indicate that alpha-synucleinopathy in tg mice is associated with oxidative stress and mitochondrial abnormalities. Because both mitochondrial abnormalities and oxidative stress are implicated in the pathogenesis of PD and other a-synucleinopathies, we will examine the pathological relationships between mitochondrial dysfunction, oxidative stress and alpha-synucleinopathies in Hua-Syn Tg mice and in cell models (Aims 1 and 2). In addition, our studies show that alpha-synuclein transgenic mice exhibit increased vulnerability to neurodegeneration induced by the chronic MPTP treatment. Thus, there could be a direct pathologic link between alpha-synuclein, mitochondrial dysfunction via environmental agents (such as pesticides), and neurodegeneration. We will use pesticides that are known to inhibit complex I activity to define the pathological interactions between these factors that known risk factors for PD using cell culture (Aim 3) and transgenic mouse models (Aim 4). These studies will provide in vivo experimental tests of processes that are directly relevant to the pathogenesis of human alpha-synucleinopathies and may lead to new therapeutic approaches. PUBLIC HEALTH RELEVANCE: Parkinson's disease and related alpha-synucleinopathies are fatal neurodegenerative diseases affecting ~1,000,000 individuals annually in US. Studies indicate that PD is associated with alpha-synuclein abnormalities, mitochondrial dysfunction, and pesticide exposure. We will determine how intrinsic (alpha- synuclein) and extrinsic (environmental toxins) factors act in concert of pathologically affect mitochondrial function and lead to neurodegeneration. Our finding will lead to better understanding of PD and new targets for therapeutic intervention.

DESCRIPTION (provided by applicant): The clinical syndrome of AD is associated with A?-deposition and the dysfunction/death of populations of neurons in a variety of neural circuits and in multiple brain regions. While A?-production and deposition are generally thought to be essential early pathogenic events AD, in vivo relationship between A? and neurodegeneration is not well understood. While transgenic (Tg) mouse models of cerebral A? deposition have been important resources for studies relevant to AD pathogenesis, these Tg mouse models only exhibit modest degeneration of cortical and hippocampal neurons even with significant A? pathology. Thus, while progressive neurodegeneration and A?-deposition is a key feature of AD in humans, the in vivo mechanisms by which A?-deposition cause neurodegeneration remain elusive. We found that, in the APPswe/PS1?E9 Tg mice, A?-deposition is associated with degeneration of monoaminergic (MAergic) systems. Both 5-HT and TH+ fibers are lost from cortical and hippocampal regions with A?-deposition in the APPswe/PS1?E9 Tg mice. Significantly, progressive degeneration of MAergic fibers is followed by the degeneration of 5-HT and NA neurons in the brain stem. Thus, APPswe/PS1?E9 mice recapitulate the profound degeneration of 5-HT and NA systems seen in human AD. The loss of these neurons and their respective axonal projections play significant roles in AD-associated dysfunctions related to learning, memory, and affect. Our finding show that A?-deposition is sufficient to cause neurodegeneration and allows for further mechanistic analysis and testing of neuroprotective strategies. In this proposal, we will define in vivo pathologic relationship between A?- accumulation/deposition and neurodegeneration. We propose the following aims: 1) Determine the onset and selectivity of monoaminergic neurodegeneration in APPswe/PS1?E9 Tg mice. 2) Characterize monoaminergic neurodegeneration in the tTA/APP Tg mice. 3) Determine the causal relationship between brain A? production/accumulation and progressive degeneration of monoaminergic neurons. 4) Determine the relationship between tau expression and MAergic neurodegeneration. 5) Determine if the monoaminergic neurodegeneration (5-HT and/or NA) contributes to progression of A? pathology in APPswe/PS1?E9 Tg mice. PUBLIC HEALTH RELEVANCE: While amyloid pathology and neurodegeneration are key features of Alzheimer's Disease (AD), pathologic relationship between amyloid pathology and neurodegeneration is currently unclear. We will exploit our newly found neurodegeneration of monoaminergic neurons in transgenic mouse models of AD to determine the pathologic relationships between amyloid pathology and neurodegeneration in brain. Since amyloid pathology is a major target of AD therapeutics and because there is an ongoing neurodegeneration at diagnosis of AD, the proposed studies will have significant impact on understanding AD pathogenesis and on therapeutic approaches for AD.

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a common neurodegenerative disease where the causes of the disease are unknown for ~90% of cases. However, a variety of genetic mutations cause PD in ~10% cases. Among the genetic causes of PD, autosomal dominant forms of familial PD (FPD) share many of the pathological and clinical features with more common late onset sporadic PD. Recently, mutations in LRRK2 gene were shown to cause late-onset FPD with variable penetrance and a-synuclein (a-Syn) pathology (a-synucleinopathy). Thus, PD caused by LRRK2 may involve interactions with environmental and genetic factors. To understand the pathogenic involvement of LRRK2 in causing PD with a-synucleinopathy, we have generated conditional human LRRK2 (hLRRK2) transgenic (Tg) mouse model where high-levels of mutant (R1441C and G2019S) and wild type (WT) hLRRK2 expression can be achieved in subcortical regions. We will determine that when expressed in subcortical neurons, mutant hLRRK2 causes progressive neuropathology in mice, including the loss of dopaminergic neurons. Because only ~50% of mutant hLRRK2 carriers develop PD within 70 years of age, mutant hLRRK2 may combine with other factors to fully express the pathogenic potential. We will test this hypothesis by testing whether mutant hLRRK2 increases vulnerability in dopaminergic neurons to dopaminergic toxin. Further, we will determine whether there is a pathologic interaction between mutant hLRRK2 and a-Syn in causing neurodegeneration of PD relevant neuronal populations.

DESCRIPTION (provided by applicant): Alpha-synuclein abnormalities are implicated in a number of neurodegenerative diseases; including Parkinson's disease (PD), Lewy Body Dementia (LBD), and Multiple Systems Atrophy (MSA). Because alpha-synuclein aggregates in neurons (PD, LBD) and/or oligodendrocytes (MSA) are prominent pathological features of these diseases, they are categorized as alpha-synucleinopathies. Collectively, alpha-synucleinopathies represent the second most common late-onset neurodegenerative disease, next to Alzheimer's disease (AD). While there are no effective therapies that can slow or stop the progression of neurodegeneration associated with alpha-synucleinopathies, availability of multiple transgenic mouse models of various alpha-synucleinopathies allows us to better understand the genesis of alpha-synuclein abnormalities in vivo and mechanisms of neurodegeneration in brain. These efforts will likely lead to identifying novel therapeutic targets for alpha-synucleinopathies. Presence of intracellular alpha-synuclein aggregates in alpha-synucleinopathy suggest that some aspect of protein degradation/quality control is dysfunctional in the diseases. Consistent with this view, we found that neurodegeneration in cellular and transgenic mouse models of neuronal alpha-synucleinopathy is associated with chronic Endoplasmic Plasmic Reticulum Stress (ERS) with abnormal Unfolded Protein Response (UPR). Our studies indicate that ERS is initiated by translocation and aggregation of alpha-synuclein within the ER. More important, pharmacological treatment with an anti-ERS compound, Salubrinal, significantly delays disease manifestation in rodent model of neuronal alpha-synucleinopathy. These results suggest that ERS response pathway, particularly modulation of phospho-eIF2alpha levels could represent a novel therapeutic target for PD and other alpha-synucleinopathies. However, because of compounds such as Salubrinal may have unknown off-target effects in vivo, a rigorous validation the phospho-eIF2alpha as therapeutic target at molecular levels are needed. Further, it is not clear if all alpha-synucleinopathies shar common neurodegenerative mechanisms. With these issues in mind, we propose following studies. First, we will study whether chronic ERS is a general feature of alpha-synucleinopathy by studying ERS in both neuronal and glial alpha-synucleinopathies (PD, LBD, MSA). Second, we will determine if aging related factors, such as oxidative stress/mitochondrial dysfunction, promotes ER accumulation of alpha-synuclein oligomers. Finally, we will determine whether the genetic alterations in components of the Perk/eIF2alpha arm of the ERS have predictable effects on alpha-synuclein dependent neurodegeneration. These studies will establish the value of ER stress pathway, particularly Perk/eIF2alpha components, as targets for development of novel therapies for PD.

? DESCRIPTION (provided by applicant): PD is a progressive neurodegenerative disease involving a variety of neuronal population. Other then symptomatic therapies, there are no ways to stop the progression of underlying neurodegeneration in PD. Unfortunately, other then symptomatic therapies, there are no ways to stop the progression of underlying neurodegeneration in PD. Currently, abnormalities in ?-synuclein (?S) is considered as a critical pathogenic agent in PD and other related diseases classified as ?-synucleinopathies. Thus, understanding how ?S abnormalities occur and cause neurodegeneration in brain appears critical for development of disease modifying therapies for PD. To understand the how ?-synucleinopathy leads to neurodegeneration, we are studying a transgenic (Tg) mouse model where the expression of the A53T mutant human ?S (Hu??S) leads to adult-onset fatal neurodegenerative disease. The affected mice exhibit many features of human ?- synucleinopathies, including ?S aggregation and neurodegeneration of multiple neuronal population. Our studies show that a stress activated kinase, c-Abl, is activated with the disease in the Hu??S(A53T) Tg mice. We propose that activation of c-Abl contributes to neurodegeneration in PD by activation of p53 and inhibition of autophagy. Specifically, we propose that c-Abl activation leads to inhibition of mdm2 and abnormal activation of cytosolic p53. Significantly, in addition to the established role of p53 in promoting apoptosis, abnormal metabolism of p53 can also inhibit autophagy. Thus, inhibitors of c-Abl may be used to attenuate the progressive neurodegeneration caused by ?S pathology. Given the therapeutic implications for multiple neurodegenerative diseases, we propose following aims to fully define the role of c-Abl activation in ?-synucleinopathy. 1) Determine the pathologic specificity of c-Al in ?-synucleinopathy using c-Abl knockout mice; 2) Determine whether mdm2/p53 pathway is involved in ?-synucleinopathy and regulation of autophagy; and 3) Determine the role of IRE1? and mTOR function in the regulation of autophagy by c-Abl/p53. By using genetic models, results of the proposed studies will provide unambiguous test of c-Abl as a therapeutic target for PD and other ?-synucleinopathies. Further, our results will provide a novel mechanistic link between c-Abl, p53, autophagy, and ?-synucleinopathy in vivo.

Summary/Abstract: Parkinson?s disease (PD) is the second most common late-onset neurodegenerative disease with the largest relative increase in mortality rates among all neurological disorders. PD is traditionally considered a motor disorder, characterized by the loss of dopaminergic neurons of the SNpc, and the presence of fibrillar cytoplasmic inclusions called Lewy bodies and Lewy neurites. However, a more global perspective on the PD is developing, motivated by pathological and clinical findings that extend beyond the basal ganglia. In particular, the majority of PD patients meet criteria for a secondary diagnosis of mild cognitive impairment that progresses dementia, a significant contributor to disease morbidity and mortality. The emerging view is that the abnormalities in ?-synuclein (?S) may be responsible for motor and non-motor symptoms in PD and Dementia with Lewy Bodies (DLB). Significantly, we recently found that abnormal ?S can cause post-synaptic deficits in vitro and in vivo via a microtubule associated protein tau (MAPT) dependent mechanism. In this proposal, we will directly determine the following hypothesis: 1) Pathogenic ?S species produce cognitive decline tau-dependent post-synaptic mechanisms and 2) MAPT-dependent postsynaptic deficits caused by exogenous ?S fibrils/oligomers contribute to cognitive deficits in sporadic PD and DLB. To determine the mechanistic basis for cognitive deficits in ?-synucleinopathy, we propose following aims: 1) Determine whether tau is required for ?S dependent synaptic and cognitive deficits; 2) Determine if mutant ?S-dependent AMPAR deficits and memory deficits are caused by multiple pathways; 3) Determine whether hippocampal ?S pathology and somatodendritic tau mislocalization correlates with dementia in PD; 4) Determine if exogenous pathogenic ?S induces pre- and/or post-synaptic deficits; and 5) Determine if pathogenic ?S induces defects in synaptic plasticity and memory in a tau dependent manner.

Summary/Abstract We propose to conduct rigorous evaluation of the role AMP-activated protein kinase (AMPK) plays in the development of Alzheimer?s Disease (AD) and provide compelling evidence to justify activation of AMPK as a therapeutic strategy for AD. AMPK is a highly conserved serine/threonine protein kinase that is activated by elevated AMP/ATP ratio. AMPK activates a host of integrated physiological responses including stimulation of energy production and mitochondrial biogenesis, activation of anti-oxidant response, and induction of autophagy via inhibition of mTOR. Since of these processes are dysfunctional in AD, the loss of AMPK activity could contribute AD pathogenesis and activation of AMPK could be beneficial. However, experiments that target AMPK in various animal models of AD have yielded ambiguous outcomes underscoring the importance of rigorous evaluation of the pathological significance of AMPK in AD. We demonstrated that partial inhibition of mitochondrial Complex I (MCI) activity with small molecules (CP2) developed in Dr. Trushina laboratory induces stress resilience pathways including AMPK activation and enhancement of mitochondrial biogenesis and cellular energetics. Importantly, collaborative efforts between Drs. Lee and Trushina have shown that CP2 treatment can attenuate amyloid dependent neurodegeneration in the Tg2576/PS1 mouse model of AD. While forebrain neurodegeneration is not a robust feature of most Tg mouse models of AD, we found that cerebral A? deposition in AD mouse models are associated with progressive degeneration of monoaminergic (MAergic) neurons, recapitulating the early degeneration of serotonergic (5-HT) and noradrenergic (NA) neurons seen in human AD patients. Using the state of art mouse models and CP2, we propose to (a) determine whether AMPK activity regulates onset and progression of AD pathology in vivo, and (b) establish to what extent loss of AMPK in neurons vs. astrocytes modulate the AD development, (c) examine whether therapeutic effect of CP2, particularly the progressive neurodegeneration, is AMPK-dependent and is facilitated in neurons or astrocytes. We will develop multiple mouse models with conditional knock out of regulatory ?1 and ?2 AMPK subunits in the whole brain using the nestin-Cre as a driver; specifically in astrocytes using pGFAP-CreERT or in neurons using SLICK- H mice (pThy1-CreERT). We will examine if constitutive loss of AMPK in brain of AP/PS Tg mouse model exacerbates AD pathology including MAergic neurodegeneration. We will also determine if the loss of AMPK directly impacts mitochondrial biology and bioenergetics in the AP/PS model. The proposed studies are based on strong premise and will provide rigorous test of the hypothesis using innovative cell biology, biochemistry and imaging techniques, and genetic interventions. The outcomes will provide the critical evidence to justify AMPK as a therapeutic target for AD.

PROJECT SUMMARY Lewy body dementias (LBD), including Parkinson?s disease dementia (PDD) and dementia with Lewy bodies (DLB), are the second most common type of degenerative dementia in the world. Unfortunately, there are currently no therapies to slow or halt the progression of LBD. Seizures associated with LBD deserve more attention because, despite the harmful impact on the patients, seizure activity can go unrecognized and untreated. Seizures or myoclonus occur in over half of DLB patients, and these symptoms hasten cognitive decline. Our preliminary studies indicate that abnormal ?-synuclein (?S), a key component of Lewy bodies, causes cognitive deficits preceded by epileptic activity. We also show that ?S-dependent synaptic and cognitive deficits and epileptic activity require endogenous tau expression. This is reminiscent of data showing that the tau-dependence of cognitive deficits and epileptic activity in the mouse models of Alzheimer?s disease (AD). Preventing epileptic activity with antiseizure drugs improves memory in models of AD, and antiseizure drugs are currently in early phase clinical trials for AD. However, antiseizure drugs have not been well investigated for ?- synucleinopathy. To better define the role of seizure activity in LBD, the following aims are proposed: Aim 1, Determine the causal relationship between the epileptiform activity and ?S-dependent cognitive deficits; Aim 2, Determine if ?S fibril inoculation model of PDD/DLB causes tau-dependent cognitive deficits mediated by epileptiform activity; and Aim 3, Define the circuitry and cellular signaling mechanisms contributing to epileptiform activity in mutant ?S models. The results of this investigation could lead to new strategies, such as antiseizure drugs and reducing tau levels, as therapies for LBD.