Faculty Bibliography

Presenilin 1 (PS1) deletion or Alzheimer's disease (AD)-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit, causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in Presenilin 1 knockout (PS1KO) cells induces abnormal Ca(2+) efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca(2+). In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca(2+) homeostasis, but correcting lysosomal Ca(2+) deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss-of-function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca(2+) homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism.

Synaptic roles for neurofilament (NF) proteins have rarely been considered. Here, we establish all four NF subunits as integral resident proteins of synapses. Compared with the population in axons, NF subunits isolated from synapses have distinctive stoichiometry and phosphorylation state, and respond differently to perturbations in vivo. Completely eliminating NF proteins from brain by genetically deleting three subunits (alpha-internexin, NFH and NFL) markedly depresses hippocampal long-term potentiation induction without detectably altering synapse morphology. Deletion of NFM in mice, but not the deletion of any other NF subunit, amplifies dopamine D1-receptor-mediated motor responses to cocaine while redistributing postsynaptic D1-receptors from endosomes to plasma membrane, consistent with a specific modulatory role of NFM in D1-receptor recycling. These results identify a distinct pool of synaptic NF subunits and establish their key role in neurotransmission in vivo, suggesting potential novel influences of NF proteins in psychiatric as well as neurological states.

In the United States, estimates indicate there are between 250,000 and 400,000 individuals with Down syndrome (DS), and nearly all will develop Alzheimer's disease (AD) pathology starting in their 30s. With the current lifespan being 55 to 60 years, approximately 70% will develop dementia, and if their life expectancy continues to increase, the number of individuals developing AD will concomitantly increase. Pathogenic and mechanistic links between DS and Alzheimer's prompted the Alzheimer's Association to partner with the Linda Crnic Institute for Down Syndrome and the Global Down Syndrome Foundation at a workshop of AD and DS experts to discuss similarities and differences, challenges, and future directions for this field. The workshop articulated a set of research priorities: (1) target identification and drug development, (2) clinical and pathological staging, (3) cognitive assessment and clinical trials, and (4) partnerships and collaborations with the ultimate goal to deliver effective disease-modifying treatments.

Over recent years, accumulated evidence suggests that autophagy induction is protective in animal models of a number of neurodegenerative diseases. Intense research in the field has elucidated different pathways through which autophagy can be upregulated and it is important to establish how modulation of these pathways impacts upon disease progression in vivo and therefore which, if any, may have further therapeutic relevance. In addition, it is important to understand how alterations in these target pathways may affect normal physiology when constitutively modulated over a long time period, as would be required for treatment of neurodegenerative diseases. Here we evaluate the potential protective effect of downregulation of calpains. We demonstrate, in Drosophila, that calpain knockdown protects against the aggregation and toxicity of proteins, like mutant huntingtin, in an autophagy-dependent fashion. Furthermore, we demonstrate that, overexpression of the calpain inhibitor, calpastatin, increases autophagosome levels and is protective in a mouse model of Huntington's disease, improving motor signs and delaying the onset of tremors. Importantly, long-term inhibition of calpains did not result in any overt deleterious phenotypes in mice. Thus, calpain inhibition, or activation of autophagy pathways downstream of calpains, may be suitable therapeutic targets for diseases like Huntington's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder which is the most common cause of dementia in the elderly today. One of the earliest symptoms of AD is olfactory dysfunction. The present study investigated the effects of amyloid beta precursor protein (AbetaPP) metabolites, including amyloid-beta (Abeta) and AbetaPP C-terminal fragments (CTF), on olfactory processing in the lateral entorhinal cortex (LEC) using the Tg2576 mouse model of human AbetaPP over-expression. The entorhinal cortex is an early target of AD related neuropathology, and the LEC plays an important role in fine odor discrimination and memory. Cohorts of transgenic and age-matched wild-type (WT) mice at 3, 6, and 16months of age (MO) were anesthetized and acute, single-unit electrophysiology was performed in the LEC. Results showed that Tg2576 exhibited early LEC hyperactivity at 3 and 6MO compared to WT mice in both local field potential and single-unit spontaneous activity. However, LEC single-unit odor responses and odor receptive fields showed no detectable difference compared to WT at any age. Finally, the very early emergence of olfactory system hyper-excitability corresponded not to detectable Abeta deposition in the olfactory system, but rather to high levels of intracellular AbetaPP-CTF and soluble Abeta in the anterior piriform cortex (aPCX), a major afferent input to the LEC, by 3MO. The present results add to the growing evidence of AbetaPP-related hyper-excitability, and further implicate both soluble Abeta and non-Abeta AbetaPP metabolites in its early emergence.

The axonal cytoskeleton of neurofilament (NF) is a long-lived network of fibrous elements believed to be a stationary structure maintained by a small pool of transported cytoskeletal precursors. Accordingly, it may be predicted that NF content in axons can vary independently from the transport rate of NF. In the present report, we confirm this prediction by showing that human NFH transgenic mice and transgenic mice expressing human NFL Ser55 (Asp) develop nearly identical abnormal patterns of NF accumulation and distribution in association with opposite changes in NF slow transport rates. We also show that the rate of NF transport in wild-type mice remains constant along a length of the optic axon where NF content varies 3-fold. Moreover, knockout mice lacking NFH develop even more extreme (6-fold) proximal to distal variation in NF number, which is associated with a normal wild-type rate of NF transport. The independence of regional NF content and NF transport is consistent with previous evidence suggesting that the rate of incorporation of transported NF precursors into a metabolically stable stationary cytoskeletal network is the major determinant of axonal NF content, enabling the generation of the striking local variations in NF number seen along axons.

Autophagy, the major lysosomal pathway for the turnover of intracellular organelles is markedly impaired in neurons in Alzheimer's disease and Alzheimer mouse models. We have previously reported that severe lysosomal and amyloid neuropathology and associated cognitive deficits in the TgCRND8 Alzheimer mouse model can be ameliorated by restoring lysosomal proteolytic capacity and autophagy flux via genetic deletion of the lysosomal protease inhibitor, cystatin B. Here we present evidence that macroautophagy is a significant pathway for lipid turnover, which is defective in TgCRND8 brain where lipids accumulate as membranous structures and lipid droplets within giant neuronal autolysosomes. Levels of multiple lipid species including several sphingolipids (ceramide, ganglioside GM3, GM2, GM1, GD3 and GD1a), cardiolipin, cholesterol and cholesteryl esters are elevated in autophagic vacuole fractions and lysosomes isolated from TgCRND8 brain. Lipids are localized in autophagosomes and autolysosomes by double immunofluorescence analyses in wild-type mice and colocalization is increased in TgCRND8 mice where abnormally abundant GM2 ganglioside-positive granules are detected in neuronal lysosomes. Cystatin B deletion in TgCRND8 significantly reduces the number of GM2-positive granules and lowers the levels of GM2 and GM3 in lysosomes, decreases lipofuscin-related autofluorescence, and eliminates giant lipid-containing autolysosomes while increasing numbers of normal-sized autolysosomes/lysosomes with reduced content of undigested components. These findings have identified macroautophagy as a previously unappreciated route for delivering membrane lipids to lysosomes for turnover, a function that has so far been considered to be mediated exclusively through the endocytic pathway, and revealed that autophagic-lysosomal dysfunction in TgCRND8 brain impedes lysosomal turnover of lipids as well as proteins. The amelioration of lipid accumulation in TgCRND8 by removing cystatin B inhibition on lysosomal proteases suggests that enhancing lysosomal proteolysis improves the overall environment of the lysosome and its clearance functions, which may be possibly relevant to a broader range of lysosomal disorders beyond Alzheimer's disease.

With increasing numbers of people with Alzheimer's and other dementias across the globe, many countries have developed national plans to deal with the resulting challenges. In the United States, the National Alzheimer's Project Act, signed into law in 2011, required the creation of such a plan with annual updates thereafter. Pursuant to this, the US Department of Health and Human Services (HHS) released the National Plan to Address Alzheimer's Disease in 2012, including an ambitious research goal of preventing and effectively treating Alzheimer's disease by 2025. To guide investments, activities, and the measurement of progress toward achieving this 2025 goal, in its first annual plan update (2013) HHS also incorporated into the plan a set of short, medium and long-term milestones. HHS further committed to updating these milestones on an ongoing basis to account for progress and setbacks, and emerging opportunities and obstacles. To assist HHS as it updates these milestones, the Alzheimer's Association convened a National Plan Milestone Workgroup consisting of scientific experts representing all areas of Alzheimer's and dementia research. The workgroup evaluated each milestone and made recommendations to ensure that they collectively constitute an adequate work plan for reaching the goal of preventing and effectively treating Alzheimer's by 2025. This report presents these Workgroup recommendations.

Defective autophagy in Alzheimer's disease (AD) promotes disease progression in diverse ways. Here, we demonstrate impaired autophagy flux in primary glial cells derived from CRND8 mice that overexpress mutant amyloid precursor protein (APP). Functionalized single-walled carbon nanotubes (SWNT) restored normal autophagy by reversing abnormal activation of mTOR signaling and deficits in lysosomal proteolysis, thereby facilitating elimination of autophagic substrates. These findings suggest SWNT as a novel neuroprotective approach to AD therapy.