Loss of proteostasis underlies the basis of multiple age-related degenerative disorders. Chaperone-mediated autophagy (CMA) activity, essential in the cellular defense against proteotoxicity, declines with age.
Maintenance of proteostasis is essential for normal cellular function and for adaptation to the always changing extracellular environment. Chaperones and the proteolytic systems are the major components of the proteostasis network. Gradual loss in functionality of some of these proteostasis pathways with age has been proposed to accelerate the course of degenerative conditions that afflict the elderly.
All cells rely on intracellular surveillance systems to maintain their proteome's homeostasis (proteostasis). These systems are especially important in neurons that, due to their postmitotic status, are highly sensitive to proteotoxic insult. Decreased neuronal protein quality control with age increases the risk of neurodegenerative diseases. In fact, presence of protein aggregates is a common feature in neurodegenerative patient brains. Interestingly, most elderly brains also display protein aggregation, even in absence of disease.
Defective autophagy, one of the components of the proteostasis network, associates with neurodegenerative diseases, including Parkinson's disease (PD) and Alzheimer's disease (AD). Macroautophagy has been proven necessary for maintenance of neuronal proteostasis and for protection against neurodegeneration.
Maintaining proteostasis is also important in vascular cells, vascular smooth muscle cells (VSMC), and macrophages. Cardiovascular disease (CVD) is the leading underlying cause of death worldwide accounting for more than 31.5% of total deaths. The main risk factors for the development of atherosclerosis—the most common cause of CV clinical events—such as obesity, hypertension, diabetes, and aging are rising in epidemic proportions due to changes in lifestyle and the growing elderly population. In atherosclerosis, hypercholesterolemia leads to vascular endothelial dysfunction and extravasation of atherogenic lipoproteins, resulting in increased adhesion and extravasation of monocytes from the circulation to the intima which progresses to atherosclerosis.
There exists a need for a method of maintaining proteostasis in neuronal and vascular cells, including in the neuronal cells of a patient at risk for a neurodegenerative disorder and vascular and macrophage cells in a patient at risk for cardiovascular disease or having atherosclerosis. This disclosure fulfills this need and provides additional advantages.
The disclosure provides a method of preventing or slowing advancement of an age related neurodegenerative disease in a subject in need thereof, comprising identifying an early symptom or biomarker of the neurodegenerative disease in the subject, and administering a therapeutically effective amount of a CMA activator to the subject, wherein the subject is asymptomatic or is in an early symptomatic stage of the age-related neurodegenerative disease.
The disclosure also provides a method of enhancing neuronal proteostasis in a subject in need of treatment for an age-related neurodegenerative disorder, comprising administering a CMA activator to the subject, wherein administering the CMA activator enhances neuronal proteostasis in the subject.
The disclosure provides a method of increasing Lamp 2A levels in neurons of a subject in need of treatment for an age-related neurodegenerative disorder, comprising administering a CMA activator to the subject, wherein administering the CMA activator increases Lamp 2A levels in the neurons of the subject.
This disclosure provides a method of delaying the onset of a neurodegenerative disorder in a patient comprising: determining the patient has a risk factor associated with developing the neurodegenerative disorder;
The disclosure provides a method of administering an amount of a Chaperone Mediated Autophagy (CMA) activator to the patient sufficient to increase CMA activity in the excitatory and/or inhibitory neurons of the patient; and thereby delaying the onset of the neurodegenerative disorder.
The disclosure provides a method of maintaining glycolytic activity in neurons of a patient. comprising administering an amount of a Chaperone Mediated Autophagy (CMA) activator to the patient sufficient to activate CMA in the excitatory and/or inhibitory neurons of the patient; and thereby maintaining glycolytic activity in the patient's neurons.
The disclosure provides a method of reducing the level of a marker Alzheimer's Dementia (AD) pathology or slowing the increase of a marker of AD pathology in a patient diagnosed as at risk of developing AD or in a patient diagnosed as having AD, comprising
Determining a first level of the marker of AD pathology in the patient; Administering an amount of a Chaperone Mediated Autophagy (CMA) activator to the patient sufficient to activate CMA in the neurons of the patient daily for a period of at least 3 months, at least 6 months, or at least 12 months; and Determining a second level of the marker of AD pathology in the patient after the administration of the CMA activator;
Comparing the first level and the second level of the marker AD pathology; and determining whether the level of the marker of AD pathology has decreased or slowed in the patient.
This disclosure provided a method of reducing gliosis or inflammation in the brain of a patient, comprising administering an amount of a Chaperone Mediated Autophagy (CMA) activator to the patient sufficient to activate CMA in the brain of the patient; and thereby reduce gliosis or inflammation brain of the patient.
This disclosure also provides a method of increasing proteostasis and/or gliosis in neurons of a patient having a neurodegenerative disorder, comprising administering an amount of a Chaperone Mediated Autophagy (CMA) activator to the patient sufficient to activate CMA in the neurons of the patient; and thereby increasing proteostasis and/or gliosis in the neurons of the patient.
The disclosure further provides a method of preventing protein aggregation in the neurons of a patient comprising Administering an amount of a Chaperone Mediated Autophagy (CMA) activator to the patient sufficient to decrease soluble protein accumulation in the neurons of the patient.
All data are mean+s.e.m. and individual values are shown in D, E, F, G, H, and IP, . n=4-8 per timepoint/genotype. Comparisons were made using Kruskal-Wallis test followed by Dunn's post hoc test (D, F, H, I), one-way ANOVA followed by Tukey's post hoc test (E, P) or two way ANOVA followed by Sidak's post hoc test (H). *p<0.05, **p<0.005, ***p<0.0005.
(B-C) Comparative quantitative proteomics of the insoluble fractions of CKL2A−/− and CKATG7−/− mice brains. (B)Venn diagram of proteins in the insoluble fractions, and (C) network visualization of gene ontology enrichment of insoluble proteins (showing similarity between nodes in CKATG7−/− (protein catabolic processes and cell cycle) and other nodes shows similarity between nodes in CKL2A−/− mice).
(D) Extracellular acidification rates (ECAR) in primary cortical neurons from CTR and CKL2A−/− mice upon addition of (D) glycolytic properties calculated from the areas under the curve in ECAR. (E-F) Clathrin-mediated endocytosis related proteins in cortex of CTR and L2A−/− mice at 6 months. (E,F) Quantifications of representative immunobots. (G) Transferrin uptake at 10 min in differentiated neuroblastoma cell lines transduced with empty vector (Control) or shL2A construct (L2A(−)). Representative images of Transferrin (light gray) and Hoechst dub (darker gray) (left). Inset: higher magnification and quantification of transferrin uptake expressed as folds of control (Ctr) cells. n=15-25 cells per condition (right). Scale bar 20 μm. (H) Arpc2 in cortex of CTR and L2A−/− mice at 6 months. (H) Quantification of representative immunoblots. (I) Immunostaining for actin in hippocampal neurons of CTR and L2A−/− mice at 6 months. (I) quantification of representative images. Scale bars: 20 μm (G, I). Data are mean±s.e.m and individual values are shown in D, E, F, H, and I. Comparisons were made using unpaired t-test (D, E-I) *p<0.05, **p<0.005, ***p<0.0005.
Prior to setting forth the invention in detail, it may be helpful to provide definitions of certain terms to be used in this disclosure. Compounds are described using standard nomenclature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. Unless clearly contraindicated by the context each compound name includes the free acid or free base form of the compound as well as all pharmaceutically acceptable salts of the compound.
The terms “a” and “an” do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. The term “or” means “and/or”. The open-ended transitional phrase “comprising” encompasses the intermediate transitional phrase “consisting essentially of” and the close-ended phrase “consisting of.” Claims reciting one of these three transitional phrases, or with an alternate transitional phrase such as “containing” or “including” can be written with any other transitional phrase unless clearly precluded by the context or art. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included within the range and independently combinable. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein. Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.
“Pharmaceutical compositions” are compositions comprising at least one active agent, such as a compound or salt of a CMA Activator, and at least one other substance, such as a carrier. Pharmaceutical compositions optionally contain one or more additional active agents. When specified, pharmaceutical compositions meet the U.S. FDA's GMP (good manufacturing practice) standards for human or non-human drugs.
Currently, diagnosis of neurodegenerative disease such as Alzheimer's disease (AD)relies on identifying mental decline, at which point significant brain damage has been done. Similarly, Parkinson's disease (PD) is identified by symptoms such as shaking or tremors, slowness of movement (bradykinesia), stiffness or rigidity of the arms and legs, and/or balance issues (postural instability). PD is a progressive disease in which the symptoms worsen over time. The methods described herein provide for preventing or slowing advancement of an age-related neurodegenerative disease in a subject in need thereof when the subject is asymptomatic or is in an early symptomatic stage of the age-related neurodegenerative disease. Early intervention may help to prevent the progression of symptoms and delay progression to late-stage age-related neurodegenerative disease.
In an aspect, a method of preventing or slowing advancement of an age-related neurodegenerative disease in a subject in need thereof comprises identifying an early symptom or biomarker of the neurodegenerative disease in the subject, and administering a therapeutically effective amount of a CMA activator to the subject. In an aspect, the subject is asymptomatic or is in an early symptomatic stage of the age-related neurodegenerative disease.
Administering the CMA activator can reduce the progression of beta-amyloid and/or tau pathology in the subject, and/or reduce pre-existing beta-amyloid and/or tau pathology in the subject. Prior to the experiments described herein, it was not expected that CMA modulation would affect beta-amyloid and/or tau pathology. The method optionally further comprises determining the progression of beta-amyloid and/or tau pathology by positron emission tomography (PET) and/or magnetic resonance (MR) imaging. 11C-labeled Pittsburgh Compound-B ([11C]PiB), also known as 2-(4-N-[11C]methylaminophenyl)-6-hydroxybenzothiazole, [18F]Florbetapir ([18F]FBP), which is also known as 18F-AV-45 or 4-{(E)-2-[6-(2-{2-[2-(18F)Fluoroethoxy]ethoxy}ethoxy)-3-pyridinyl]vinyl}-N-methylaniline, [18F]Florbetaben ([18F]FBB), and [18F]Flutemetamol ([18F]FMT) are radiotracers for beta-amyloid {ET imaging. The PET ligand [18F]AV-1451 binds tau-positive inclusions. The levels of tau protein (total tau or phosphorylated tau) or beta-amyloid (e.g., Aβ42) in the plasma or cerebrospinal fluid (CSF) of the subject can also be used to determine the progression of beta-amyloid and/or tau pathology.
It was also unexpectedly found that CMA mdosulators have an effect on gliosis, defined herein as progression of glial cells. In an aspect, administering the CMA inhibitor reduces gliosis in the brain of the subject, for example as determined by positron emission tomography (PET) and/or magnetic resonance (MR) imaging.
Progressive subcortical gliosis is a chromosome-17-linked dementia with unique pathologic features including fibrillary astrocytosis. Early symptoms include personality and emotional changes, lack of judgment and insight, deterioration in social behavior, delusions, paranoia, auditory and visual hallucinations, and depression.
In an aspect, the method further comprises detecting an increase in neuronal glycolysis after administering the CMA activator. Unexpectedly, CMA activation has been shown to increase glycolysis.
In another aspect, a method of enhancing neuronal proteostasis in a subject in need of treatment for an age-related neurodegenerative disorder comprises administering a CMA activator to the subject, wherein administering the CMA activator enhances neuronal proteostasis in the subject. In an aspect, administering the CMA activator reduces the progression of beta-amyloid and/or tau pathology in the subject, and the method optionally comprises determining the progression of beta-amyloid and/or tau pathology by positron emission tomography (PET) and/or magnetic resonance (MR) imaging, or by tau protein (total tau or phosphorylated tau) or beta-amyloid (e.g., Aβ42) in the plasma or cerebrospinal fluid (CSF) of the subject. In an aspect, the method further comprises detecting an increase in neuronal glycolysis after administering the CMA activator.
In yet another aspect, a method of increasing Lamp 2A levels in neurons of a subject in need of treatment for an age-related neurodegenerative disorder, comprises administering a CMA activator to the subject, wherein administering the CMA activator increases Lamp 2A levels in the neurons of the subject.
In another aspect the disclosure provides a method of protecting a subject against developing atherosclerosis, significantly reducing the likelihood of a subject at risk for atherosclerosis from developing atherosclerosis, slowing the progression of atherosclerosis in a subject having atherosclerosis, or decreasing atherosclerosis in a subject having atherosclerosis by administering a therapeutically effective amount of a CMA activator to the subject. The disclosure also provides a method of protecting vascular cells, including smooth muscle vascular cells in a subject by administering a CMA activator to the subject. The disclosure also provides a method of preventing or decreasing macrophages having a pro-inflammatory phenotype, for example in a subject having atherosclerosis, include asymptomatic atherosclerosis, comprising administering a CMA activator to the subject.
Macrophages in atherosclerotic lesions actively participate in lipoprotein ingestion and accumulation giving rise to foam cells filled with lipid droplets. Accumulation of foam cells contributes to lipid storage and atherosclerotic plaque growth and chronic inflammatory conditions.
Pathologic inflammatory conditions are frequently correlated with dynamic alterations in macrophage activation, with classically activated M1 cells associated with promoting and sustaining inflammation and M2 cells implicated in resolution or smoldering chronic inflammation (DOI: 10.2174/1874467215666220324114624). Reduced CMA in macrophages promotes a bias in macrophage differentiation toward M1 (pro-inflammatory phenotype).
Compounds disclosed herein can be administered as the neat chemical, but are preferably administered as a pharmaceutical composition. Accordingly, the disclosure provides pharmaceutical compositions comprising a compound or pharmaceutically acceptable salt of a CMA modulator, such as a CMA Activator, together with at least one pharmaceutically acceptable carrier. In certain embodiments the pharmaceutical composition is in a dosage form that contains from about 0.1 mg to about 2000 mg, from about 10 mg to about 1000 mg, from about 100 mg to about 800 mg, or from about 200 mg to about 600 mg of a compound of a CMA Activator and optionally from about 0.1 mg to about 2000 mg, from about 10 mg to about 1000 mg, from about 100 mg to about 800 mg, or from about 200 mg to about 600 mg of an additional active agent in a unit dosage form.
Compounds disclosed herein may be administered orally, topically, parenterally, by inhalation or spray, sublingually, transdermally, via buccal administration, rectally, as an ophthalmic solution, through intravitreal injection or by other means, in dosage unit formulations containing conventional pharmaceutically acceptable carriers. The pharmaceutical composition may be formulated as any pharmaceutically useful form, e.g., as an aerosol, a cream, a gel, a pill, a capsule, a tablet, a syrup, a transdermal patch, or an ophthalmic solution for topical or intravitreal injection. Some dosage forms, such as tablets and capsules, are subdivided into suitably sized unit doses containing appropriate quantities of the active components, e.g., an effective amount to achieve the desired purpose.
Carriers include excipients and diluents and must be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to the patient being treated. The carrier can be inert, or it can possess pharmaceutical benefits of its own. The amount of carrier employed in conjunction with the compound is sufficient to provide a practical quantity of material for administration per unit dose of the compound.
Classes of carriers include, but are not limited to binders, buffering agents, coloring agents, diluents, disintegrants, emulsifiers, flavorants, glidants, lubricants, preservatives, stabilizers, surfactants, tableting agents, and wetting agents. Some carriers may be listed in more than one class, for example vegetable oil may be used as a lubricant in some formulations and a diluent in others. Exemplary pharmaceutically acceptable carriers include sugars, starches, celluloses, powdered tragacanth, malt, gelatin; talc, and vegetable oils. Optional active agents may be included in a pharmaceutical composition, which do not substantially interfere with the activity of the compound of the present disclosure.
The pharmaceutical compositions/combinations can be formulated for oral administration. These compositions contain between 0.1 and 99 weight % (wt. %) of a CMA Activator and usually at least about 5 wt. % of a compound of a CMA Activator. Some embodiments contain from about 25 wt. % to about 50 wt. % or from about 5 wt. % to about 75 wt. % of the compound of a CMA Activator.
The disclosure also provides methods of selectively activating chaperone-mediated autophagy (CMA) in a subject in need thereof comprising administering to the subject a CMA Activator in an amount effective to activate CMA in the subject.
The subject can have, for example, a neurodegenerative disease, such as tauopathies, (Frontotemporal Dementia, Alzheimer's disease), Parkinson's Disease, Huntington's Disease, prion diseases, amyotrophic lateral sclerosis, retinal degeneration (dry or wet macular degeneration, retinitis pigmentosa, diabetic retinopathy, glaucoma, Leber congenital amaurosis), diabetes, acute liver failure, non-alcoholic steatohepatitis (NASH), hepatosteatosis, alcoholic fatty liver, renal failure and chronic kidney disease, emphysema, sporadic inclusion body myositis, spinal cord injury, traumatic brain injury, fibrosis (liver, kidney, or lung), a lysosomal storage disorder, a cardiovascular disease, and immunosenescence. Lysosomal storage disorders include, but are not limited to, cystinosis, galactosialidosis, and mucolipidosis. The subject may also have a disease or condition in which CMA is upregulated such as cancer or Lupus. The subject can have reduced CMA compared to a normal subject prior to administering the compound. Preferably, the compound does not affect macroautophagy or other autophagic pathways. In macroautophagy, proteins and organelles are sequestered in double-membrane vesicles and delivered to lysosomes for degradation. In CMA, protein substrates are selectively identified and targeted to the lysosome via interactions with a cytosolic chaperone and cross the lysosomal membrane through a translocation complex.
The disclosure also provides a method of protecting cells from oxidative stress, hypoxia, proteotoxicity, genotoxic insults or damage and/or lipotoxicity in a subject in need thereof comprising administering to the subject any of the compounds disclosed herein, or a combination of a CMA Activator, in an amount effective to protect cells from oxidative stress, hypoxia proteotoxicity, genotoxic insults or damage, and/or lipotoxicity. The subject can have, for example, one or more of the chronic conditions that have been associated with increased oxidative stress and oxidation and a background of propensity to proteotoxicity. The cells being protected can comprise, for example, cardiac cells, kidney and liver cells, neurons and glia, myocytes, fibroblasts and/or immune cells. The compound can, for example, selectively activate chaperone-mediated autophagy (CMA). In one embodiment, the compound does not affect macroautophagy.
In a specific aspect, the subject is suffering from mild cognitive impairment. As used herein, mild cognitive impairment is the stage between the expected cognitive decline due to aging and the more serious decline of dementia. Forgetfulness, losing train of thought or difficulty following conversations, difficulty making decisions, getting lost in familiar environments and poor judgment can be signs of mild cognitive impairment. Mild cognitive impairment can progress to Alzheimer's disease or other forms of dementia.
Exemplary age-related neurodegenerative diseases include Alzheimer's disease (AD), Lewy body dementia, Parkinson's disease (PD), Huntington's disease, Amyotrophic lateral sclerosis (ALS), Frontotemporal dementia (FTD), Spinocerebellar ataxias (SCAs), Progressive subcortical gliosis, and the like.
When the age-related neurodegenerative disease is AD, the subject for the methods described herein subject may not suffer from dementia. Exemplary early symptoms of AD include memory loss and/or confusion, difficulty concentrating, difficulty completing daily tasks, time and/or place confusion, difficulty with visual images and/or spatial relationships, difficulty conversing, misplacing objects, poor judgment, withdrawal from activities, changes in mood and personality. Exemplary biomarkers for AD are tau protein (total tau or phosphorylated tau) or beta-amyloid (e.g., Aβ42) in the plasma or cerebrospinal fluid (CSF) of the subject.
In Lewy body dementia, protein deposits called Lewy bodies develop in nerve cells in the regions of the brain involved in cognition, memory and movement. Early symptoms of Lewy body dementia include loss of small, acting out while dreaming, visual hallucinations, confusion, difficulty maintaining attention, memory loss, changes in handwriting, muscle rigidity, falling, and drowsiness. Currently there are no verified biomarkers for Lewy body dementia.
PD is a progressive nervous system disorder that affects movement. Exemplary early symptoms of PD include slight tremors in the fingers, thumbs, hand or chin; small handwriting (also called micrographia); loss of smell; difficulty sleeping including sudden movements in sleep; difficulty moving or walking; constipation; a soft or low voice; facial masking; dizziness or fainting; and/or stooping, leaning or slouching while standing. Currently there are no verified clinical biomarkers for PD.
Huntington's disease is a genetic disorder that causes progressive degeneration of nerve cells in the brain. Early symptoms of Huntington's disease include difficulty concentrating, memory lapses, depression, clumsiness, small involuntary movements and mood swings. Mutant Huntington protein (mHtt) is a biomarker for Huntington's disease. Subjects who carry the Huntington mutation can be treated by the methods described herein.
ALS is a rare, progressive disease involving the nerve cells responsible for controlling voluntary movements. Early symptoms of ALS include muscle twitches in the arm, leg, shoulder or tongue; muscle cramps; stiff muscles; muscle weakness of the arm, leg, neck or diaphragm; slurred and nasal speech; and difficultly chewing or swallowing. Currently there are no validated biomarkers for ALS.
FTD, sometimes called Pick' disease, is a group of neurological disorders in which nerve cells in the front and temporal lobes of the brain are lost. Early symptoms of FTD include changes to personality and behavior and/or difficulties with language. Clinically, differentiating between FTD and AD is challenging.
Spinocerebellar ataxias (SCAs) are progressive disorders in which the cerebellum slowly degenerates, often accompanied by degenerative changes in the brainstem and other parts of the central nervous system. Early symptoms of SCAs are problems with coordination and balance, speech and swallowing difficulties, muscle stiffness, weakness of the muscles that control eye movement, and cognitive impairment. SCA1, SCA2, SCA3, SCA6, SCA7 and SCA17 share the same pathogenic mechanism of CAG trinucleotide repeat expansions encoding elongated polyglutamine tracts. There is no serum biomarker for SCAs.
In an embodiment the subject is a mammal. In certain embodiments the subject is a human, for example a human patient undergoing medical treatment. The subject may also be a companion a non-human mammal, such as a companion animal, e.g. cats and dogs, or a livestock animal.
For diagnostic or research applications, a wide variety of mammals will be suitable subjects including rodents (e.g. mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like. Additionally, for in vitro applications, such as in vitro diagnostic and research applications, body fluids (e.g., blood, plasma, serum, cellular interstitial fluid, cerebrospinal fluid, saliva, feces and urine) and cell and tissue samples of the above subjects will be suitable for use.
An effective amount of a pharmaceutical composition may be an amount sufficient to inhibit the progression of a disease or disorder, cause a regression of a disease or disorder, reduce symptoms of a disease or disorder, or significantly alter a level of a marker of a disease or disorder.
An effective amount of a compound or pharmaceutical composition described herein will also provide a sufficient concentration of a CMA Activator when administered to a subject. A sufficient concentration is a concentration of the CMA Activator in the patient's body necessary to prevent or combat a CMA mediated disease or disorder or other disease or disorder for which a CMA Activator is effective. Such an amount may be ascertained experimentally, for example by assaying blood concentration of the compound, or theoretically, by calculating bioavailability.
Methods of treatment include providing certain dosage amounts of a CMA Activator to a subject or patient. Dosage levels of each compound of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per patient per day). The amount of compound that may be combined with the carrier materials to produce a single dosage form will vary depending upon the patient treated and the particular mode of administration. Dosage unit forms will generally contain between from about 1 mg to about 500 mg of each active compound. In certain embodiments 25 mg to 500 mg, or 25 mg to 200 mg of a CMA Activator are provided daily to a patient. Frequency of dosage may also vary depending on the compound used and the particular disease treated. However, for treatment of most diseases and disorders, a dosage regimen of 4 times daily or less can be used and in certain embodiments a dosage regimen of 1 or 2 times daily is used.
It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
In an embodiment, the invention provides a method of treating a lysosomal storage disorder in a patient identified as in need of such treatment, the method comprising providing to the patient an effective amount of a CMA Activator. The CMA Activator may be administered alone as the only active agent, or in combination with one or more other active agent.
Male wild-type mice (C57BL/6J) or transgenic for CaMKIIα-Cre (B6.Cg-Tg(Camk2α-cre)T29-1Stl/J, Jackson Laboratory), LAMP-2Aflox/flox (Schneider et al., 2014), TauP301L (line pR5), Tg (APPSwe; PS2N141I; TauP301L), PS19 (TauP301S) (Yoshiyama et al., 2007) and Atg7f/f were used in the study. Conditional LAMP-2A deletion was generated by breeding LAMP-2Aflox/flox with the transgenic Cre mice of interest. Knockout for L2A in the whole body (L2A−/−) was generated by insemination of a wild-type female with spermatozoids with L2A floxed to excise this gene in all tissues in the offspring. Male littermate wild type and only L2Af/f were separately analyzed for each test and because no differences were detected among them, they were grouped in the results as “control” (CTR) for the experimental group (CamKII□ CreL2Af/f or L2A−/−). All mice were genotyped at weaning and genotyping was re-confirmed postmortem to correct for any possible misplacement during husbandry. Mice were all in the C57BL/6J background and maintained under specific pathogen-free conditions in ventilated cages with no more than 5 mice per cage. Only males were used in this study due to the complexity in generating the quadruple transgenic mouse model with L2A knock out in homozygosis (for which we took advantage of the location of the Lamp2 gene in the X chromosome). Age of the animals was 4-6 months in most experiments except when otherwise indicated in text, figures and figure legends. Animals were maintained at 19-23° C. in 12 h light/dark cycle. Mice were fed ad libitum. CA77.1 was administered as sucralose jelly pellets for a daily dose of 30 mg/Kg body weight whereas the vehicle treated group received the same sucralose jelly pellet without drug. Briefly, for preparation of the jelly pellets the final amount of the compound per day (adjusted per animal weight) was dissolved in ethanol and then mixed with a warm gelatin solution (100 mg/ml, 10 mg/ml sucralose in water), that was poured into 24 well flat bottom plates for solidification. To avoid animal stress and competition for the pellets, animals were separated with a grid spacer in the same cage that they were housed, eating of the pellet was monitored and the spacer was removed as soon as all mice consumed the pellet (average time 2 min). Sentinel animals were included in each study to determine brain exposure at the end of treatment. These animals received the same batch of jelly pellets in parallel to the experimental group. Animals were assigned randomly to the vehicle and placebo groups and no animals were eliminated from the study. All genotyping, breeding, handling and treatments in this study were done according to protocol and all animal studies were under an animal study protocol approved by the Institutional Animal Care and Use Committee of Albert Einstein College of Medicine.
Cortical neurons were obtained from control (CTR) and L2AKO PO-P1 postnatal mice and neuronal cultures were prepared as follows: brain cortices were dissected and enzymatically digested (0.36 mg/ml papain in phosphate buffered saline (PBS) with D-glucose (6 mg/ml) and 1% bovine serum albumin for 15 minutes)37° C. Neurons collected by centrifugation, were resuspended in Neurobasal Medium (ThermoFisher 10888022), supplemented with 2% B27-Supplement (Gibco-Invitrogen, 17504044), 1% Penicillin/Streptomycin and 1% GlutaMAX (Fisher, 35050-061) and plated at a density of 2.5×105 cells/cm2 into 24-well Seahorse Bioscience plates (Agilent, 100777-004) pre-coated with CELL-TAK (CORNING, 354240) or in coverslips. The first 24 h the media contained fetal calf serum 15% (v/v) heat-inactivated. Cells in coverslips were co-stained with NeuN, GFAP and Hoechst to assess level of glial presence in the primary neuronal cultures.
Mouse embryonic NIH3T3 fibroblasts from the American Type Culture Collection (ATCC) and mouse neuroblastoma CAD cell lines (gift from Dr. Duncan Wilson, Albert Einstein College of Medicine) were maintained in DMEM (Sigma-Aldrich) in the presence of 10% newborn calf serum (NCS) (Atlanta Biologicals). CAD cells were differentiated by serum removal and used at 5 days post-starvation. Lentivirus expressing the shRNA constructs against LAMP-2A and Atg7 were generated by the same protocol using the shRNA previously described.
Primary antibodies were from the following sources (dilutions, commercial source and catalog number indicated in brackets): rabbit anti-LAMP-2A (1:5000, ThermoFisher Scientific, 512200), rabbit anti-LC3 (1:1000, Cell Signaling, 2775), rabbit anti-p62 (1:1000, Enzo Life Sciences, BMLPW98600100), mouse anti-β-actin (1:10000, Sigma, A4700), rat anti-LAMP1 (1:1000, Hybridoma Bank, 1D4B), rabbit anti-4HNE (1:1000, abcam, ab46545), rabbit anti-GFAP (1:1000, Dako, Z0334), rabbit anti-GFAP (1:1000, abcam, ab5804), rabbit anti-ubiquitin (1:1000, Dako, Z0458), rabbit anti-K48-ubiquitin (1:1000, Millipore, 05-1307), rabbit anti-K63-ubiquitin (1:1000, Millipore, 05-1308), mouse anti-Aβ (1:1000, Novus Biologicals, MOAB2-AF488), mouse anti-human-Tau (1:1000, Abcam, Tau13 ab 19030), mouse anti-Tau (HT7 clone) (1:1000, ThermoFisher Scientific, MN1000), mouse anti-phosphoTau Ser202-Thr205 (AT8 clone) (1:1000, ThermoFisher Scientific, MN1020), mouse anti-phosphoTau Ser422 (1:1000, generated in house using the phosphopeptide CSIDMVD-pS-PQLATLAD as antigen (Grueninger et al., 2010)), rabbit anti-GATE16 (1:1000, MBL, PM038), mouse anti-misconformed tau (MC1) (1:1000, a generous gift from Dr. Peter Davis), rabbit anti-Clathrin (Clone D3C6—1:1000—Cell Signaling 4796S), mouse anti-AP2α (Clone 3B5—1:1000—ThermoFisher Scientific, MA1-872), rabbit anti-Arpc2 (1:1000—Novus Biological NBP188852), mouse anti-APP (1:1000—Biolegend 802803), mouse anti-PDH (1:1000—ThermoFisher Scientific, 459400), rabbit anti-CathepsinD (CathD—1:1000—Abcam #ab75852), rabbit anti-GBA (1:1000—Sigma Aldrich, G4171), mouse anti-Hsc70 (1:1000—Novus Biological NB120-2788), rat anti-Hsp90 (1:1000—Stressgen ADI-SPA-835-F), rabbit anti-Hsp40 (1:1000—Stressgen ADI-SPA-400), mouse anti-Rac1 (Clone23A8—1:1000—Millipore 05-389), rabbit anti-Phlpp1 (1:1000—Bethyl A300-660A), anti-Rictor (1:1000—Bethyl A300-459A), anti-CathepsinA (Ctsa—1:1000—Abcam #ab184553), rabbit anti-RARα (1:1000—Cell Signaling 2554), mouse anti-CaMKIIα (1:200—Millipore #05-532). All the secondary antibodies were from ThermoFisher Scientific. All antibodies used in this study were from commercial sources and were validated following the multiple dilution method and, where available, using cell lines or tissues from animals knock-out for the antigen.
All behavioral procedures were performed by investigators blind to genotype of each group or nature of intervention. To decrease stress related to procedures, all animals were first habituated to handling by the experimenter and to the procedure room for at least 1 hour prior to testing. Limb Clasping. Clasping was assessed for 5 seconds and scored. Spontaneous alternation in a Y-maze. Mice were allowed to freely explore the maze for 10 min. Number and order of arm entries were quantified. Alternation index was calculated as. Stride length. Stride length was measured in a 4.5 cm×40 cm corridor following inking of hindlimb paws. The three longest stride lengths (corresponding to maximum velocity) were considered. Short-term memory test in Y-maze. Test was performed in a Y-shaped maze with three arms angled at 120°. Visual cues were placed on surrounding walls. On the first trial (learning), animals explored the maze for 8 minutes with only two arms opened (‘start’ and ‘familiar’ arms). Access to the third arm (‘novel’) was blocked by an opaque door. After a 1 hour retention time, mice were placed again in the maze for 5 minutes with all arms accessible (test). Exploration was recorded and an automated tracking system was used. Data are reported as fraction of time spent in the novel arm. Negative geotaxis. Mice are placed on the sloped platform (50°) facing in a downward direction. The latency to turn and orient themselves to be facing up the slope was recorded. Novel object recognition. Novel object recognition was performed after training mice in an open field arena with identical objects for 4 minutes, followed by 2 hours retention time. Mice were placed in the same arena after replacing one of the familiar objects by a novel object and exploration of both objects was quantified for 4 minutes. Novelty preference is quantified as amount of time dedicated to the exploration of the novel object. Discrimination index is the difference between the exploration time of the novel and familiar object over the total exploration time. Elevated plus maze. Anxiety-like behavior was quantified as follows. Briefly, mice were allowed to freely explore an elevated plus maze with two open arms and two closed arms. Quantification of the % of time spent to explore the open arms versus the closed arms was done. Forced Swim test. Mice were placed in cylinder tank (30 cm×20 cm) filled with water at room temperature. Animals were gently placed in water and immobility was quantified over a total time of 9 minutes. Open field. Mice were allowed to freely move in an open field arena (50×50 cm) for 10 min. Tracking was performed using ezTrack (Pennington et al., 2019). The number of animals selected for each behavior test was determined by power analysis. In those cases, in which test allowed for repetition without risking a co-funding effect of “learning the test” or where multiple tests in the same animal were possible, we performed testing in higher number of animals than the minimal determined by power analysis in order to further strengthen confidence in the findings.
Mice were euthanized with pentobarbital overdose (100 mg/kg i.p.) and intracardially perfused with 0.9% saline solution. Brains were removed quickly after death. Each brain was then dissected along the midline. The right hemisphere was post-fixed overnight in 4% paraformaldehyde, cryoprotected in PBS containing 20% sucrose before being freeze by immersion in a cold isopentane bath (−50° C.), and stored immediately at −80° C. until sectioning. Brains were sectioned in a Leica CM3050S cryostat (Leica Microsystem, Wetzlar, Germany) at −20° C. in either coronal or sagittal 40 μm-thick free-floating sections and stored in PBS containing 0.2% sodium azide at 4° C. until use. The left hemisphere was dissected, and several brain regions were collected for further analysis: cortex, hippocampus, midbrain, striatum and cerebellum. Samples were stored at −80° C. until use. Prior to staining, sections of appropriate levels (e.g. striatum, midbrain or hippocampus) were selected. Immunostainings were performed as follows. Briefly, selected sections were washed, incubated in blocking buffer, and then incubated overnight with the appropriate primary antibody. The following day, sections were washed 3 times for 5 minutes in PBS, incubated with the appropriate anti-specie secondary antibody (1:2000), washed 3 times in PBS and mounted. Cell nuclei were stained using Hoechst (Life Technologies, 33342) at 1:5,000 for 30 sec prior to mounting. ProLong Diamond mounting medium was used (ThermoFisher Scientific P36965). For the immunostainings in the Tg and Tg-L2A−/− immunofluorescent detection of phosphorylated tau aggregates and amyloid plaques was performed. The amyloid-specific BAP-2 antibody was replaced by MOAB2-AF488 (Novus Biologicals). Images were acquired with an Axiovert 200 fluorescence microscope (Carl Zeiss Microscopy), or when full brain sections were imaged individual images from the scanning of brain slices were mounted with an ApoTome.2 slider, or a Leica confocal TCS-SP8 (Leica Microsystem) and prepared using ImageJ Software (NIH). A perceptually uniform lookup table (Magma) was used to enhance contrast and highlight pattern and intensity differences between experimental groups.
Thioflavin S staining was performed prior to incubation in the blocking buffer using a 0.5% Thioflavin S solution (Santa Cruz, sc391005) in water for 7 minutes at room temperature.
Where indicated mouse liver, lung and kidneys were dissected and fixed in 1% PFA overnight and paraffin embedded. Tissues were sectioned, stained with hematoxylin and eosin (H&E), and analyzed by an expert pathologist, blind to the treatment groups, to score for possible presence of toxicity in these organs. Blood cell count in the groups of mice administered vehicle or CA was analyzed in tail blood drawn monthly and at the moment of tissue dissection using an Oxford Science Forcyte Blood Analysis Unit.
Protein concentration was determined using the Lowry method with bovine serum albumin as a standard (Lowry et al., 1951). Dissected brain regions were solubilized on ice with RIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15M NaCl, 0.01M sodium phosphate, pH7.2) followed by sonication. Immunoblotting was performed after transferring SDS-PAGE gels to nitrocellulose membrane and blocking with 5% low-fat in milk 0.01% Tween-TBS for 1 h at room temperature. The proteins of interest were visualized after incubation with primaries by chemiluminescence using peroxidase-conjugated secondary antibodies in LAS-3000 Imaging System (Fujifilm, Tokyo, Japan). Densitometric quantification of the immunoblotted membranes was performed using ImageJ (NIH). All protein quantifications were done upon normalization of protein levels to a loading control (β-actin) or Ponceau staining and expressed as fold of the relevant control group.
Macroautophagic flux was measured in protein lysates using immunoblot for LC3-II in cells treated or not with lysosomal protease inhibitors (20 mM ammonium chloride and 100 μM leupeptin). Flux was calculated as the increase in levels of LC3-II in protease inhibitors-treated cells relative to untreated cells. CMA activity was measured in cells stably transduced with lentivirus carrying the KFERQ-PS-Dendra reporter and plated in glass-bottom 96-well. Sixteen hours after photo switching with a LED lamp (405 nm for 3 minutes), cells were fixed with 4% PFA and imaged using high-content microscopy (Operetta system, Perkin Elmer). Images were quantified using the manufacturer's software in a minimum of 800 cells.
Transferrin internalization was performed as previously. Briefly, CAD cells grown in serum-free DMEM were incubated for 10min using Alexa555-conjugated transferrin (25 μg/ml; Life Technologies). The cells were then transferred on ice and wash 3 times with ice-cold PBS. Cells were then fixed for immunofluorescence.
Brain homogenates were prepared as described in. Homogenates from several mice of each genotype were pooled and diluted to a final protein concentration of 1 mg/ml. Sarkosyl was then added to a final concentration of 1% and the homogenates incubated for 30 min at 4° C. The homogenates were subsequently centrifuged at 100,000× g for 1 hr. Pelleted proteins were sent for proteomic analysis or were resuspended directly in SDS-PAGE sample buffer and boiled for 2 min. For each genotype, equal volumes of resuspended pellet were used for SDS-PAGE/western blotting.
Tau, pS422-Tau, pS202/T205-Tau, and aggregated tau were measured by immunoassay in brain extracts as described in (Grueninger et al., 2010), except that the MSD assay format was replaced by AlphaLISA immunoassay technology (Perkin-Elmer). Aβ42 Enzyme-Linked Immunosorbent Assay (ELISA)
Aβ42 levels were measured by ELISA using a commercial kit (ThermoFisher #KHB3442). Mouse brain lysates were diluted 1:50 in the provided diluent and assay was performed following manufacturer's recommendations.
Oxygen consumption rates and extracellular acidification rates were measured using a 24-well Seahorse Bioanalyzer XF 24 according to the manufacturer's instructions (Agilent Technologies). Briefly, neurons were plated into 24-well plates pre-coated with CELL-TAK (CORNING, 354240at a concentration of 1.8×1014 cells/well and used at 14 days-in-vitro. Once in the reader, plates were sequentially injected 10 mM glucose, 1.0 μM oligomycin and 50 mM 2-Deoxyglucose (2-DG) in artificial cerebrospinal fluid (aCSF, 120 mM NaCl, 3.5 mM KCl, 1.3 mM CaCl2, 0.4 mM KH2PO4, 1 mM MgCl2, 5 mM HEPES) +2 mM glutamine, pH: 7.4. Differentiated CAD cells plated on gelatin-coated plates were switched to manufacture provided base medium supplemented with 2 mM L-glutamine and sequentially injected with 10 mM glucose, 2 mM oligomycin and 100 mM 2DG in the bioanalyzer. Quantifications were performed using Seahorse Wave Desktop software. Data were normalized to cell number using CyQuant (ThermoFisher, C7026).
ICR (CD-1) male mice were fasted at least three hours and water was available ad libitum before the study. Animals were housed in a controlled environment, target conditions: temperature 18 to 29° C., relative humidity 30 to 70%. Temperature and relative humidity were monitored daily. An electronic time-controlled lighting system was used to provide a 12 hr light/12 hr dark cycle. 3 mice for each indicated time point were administered 10 mg/Kg CA77.1 by oral gavage or 1 mg/Kg by intravenous injection using 30% PEG-400, 65% D5W (5% dextrose in water), 5% Tween-80 vehicle. Mice were sacrificed, and brain samples were harvested at 0 hr, 0.25 hr, 0.5 hr, 1 hr, 2 hr, 4 hr, 8 hr, 24 hr, and analyzed for CA77.1 levels using LC-MS/MS. Pharmacokinetics parameters were calculated using Phoenix WinNonlin 6.3. Experiments performed at SIMM-SERVIER joint Biopharmacy Laboratory.
Sarkosyl-insoluble fractions from three different animals per genotype (Ctr, CKL2A−/−, and CKATG7−/−) were pooled. Quantitative proteomic analysis was performed using iTRAQ multiplex (Applied Biomics). For each protein hit the average ratio(s) for the protein, the number of peptide ratios that contributed and the geometric standard deviation were determined. Values in the experimental groups were compared to Ctr and are represented as folds. For the comparative analysis between the sarkosyl-insoluble fractions of CKL2A−/− and CKATG7−/− mice (
Protein was precipitated from lysates (cortex and hippocampus pooled) from three mice of each genotype (WT, L2A−/−, Tg, Tg-L2A−/−), solubilized in 8M urea, 0.1 M ammonium bicarbonate pH 8.0, 150 mM NaCl, complete mini protease and phosphatase inhibitors (Roche) and cysteine residues were reduced and alkylated with TCEP and iodoacetamide, followed by a 5-fold dilution with 0.1M ammonium bicarbonate. Proteins were digested into peptides by the addition of trypsin over night at 37° C. (1 μg trypsin per 100 μg lysate). Samples were desalted on C18 cartridges (NEST), lyophilized, resuspended in 4% formic acid, 3% acetonitrile and approximately lug of digested peptides per sample were loaded onto a 75 μm ID column packed with 25 cm of Reprosil C18 1.9 μm, 120 Å particles (Dr. Maisch GmbH HPLC, Germany). Peptides were eluted into an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) over the course of a 120 minute acquisition by gradient elution delivered by an Easy 1200 nLC system (Thermo Fisher Scientific). The composition of mobile phase A and B were 0.1% formic acid in water and 0.1% formic acid in 80% acetonitrile, respectively. All MS spectra were collected with orbitrap detection and the most abundant ions were fragmented by higher energy collision dissociation and detected in the ion trap, with a 3 second cycle time between MS1 spectra. All data were searched against the Uniprot mouse database (downloaded Jul. 19, 2016). Peptide and protein identification searches were performed using the MaxQuant data analysis algorithm, and all peptide and protein identifications were filtered to a 1% false-discovery rate. Label-free quantification and statistical testing was performed using the MSstats statistical R-package. Significantly modified proteins were selected by p<0.05 followed correction using the Benjamini-Hochberg protocol (FDR 5%). When FDR correction led to no hit, inspection of uncorrected p-values distribution was performed: if an anti-conservative distribution was observed, we applied alternative method of false discovery rate control by lowering threshold for significance (p<0.01) and using fold change cutoff (|fold change|>1.25), as previously suggested. The mass spectrometry data files (raw and search results) have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with dataset identifier PXD017108.
Relevant protein lists were ranked according to fold change compared to Ctr mice and submitted to rank-rank hypergeometric overlap test in R (v. 3.6.2) using default settings. Colormap indicates -log10 p value of the exact Fisher test by bin. We also used proteome-wide transition mapping, that consists of serialized differential expression analysis between genotypes using overlap quantification by RRHO and has the advantage of being threshold free and allowing a statistical assessment of the overlap extent. In our studies with Tg, Tg-L2A−/− and L2A−/− mice, we used comparison with this method of a fold change-ranked list of proteins of each genotype (normalized to CTR).
Proteomic results were ranked according based on fold change and submitted to a GSEA Preranked analysis in GSEA (v. 4.0.2) with 1000 permutations. Terms smaller than 15 genes or bigger than 500 were discarded as previously reported. The enrichment map was generated in Cytoscape (3.7.1) using Enrichment map plugin (3.2.0) using the following thresholds: p value <0.05, FDR <0.001. Alternatively, proteomic results were submitted to ontology analysis using Enrichr. Node size indicates the number of proteins per node. Major clusters are circled, and the associated name represent the major functional association.
Analysis of content in KFERQ-like motif was performed using the publicly available tool: https://http://tinyurl.com/kferq.
Data set used were from (Grubman et al., Nat. Neurosci. (2019) 22: 2087-2097; Mathys et al., Nature (2019) 570: 332-337). Raw counts per cell and metadata (cell type identification and patients' information) were obtained from the Synapse portal (https://www.synapse.org/#!Synapse:syn18485175). Filtered counts provided by (Mathys et al., 2019) were used. Single cell gene expression data and metadata were obtained from http://adsn.ddnetbio.com/ for the dataset (Grubman et al., 2019) Prior to calculations, counts were log normalized using SCANPY (v.1.4.5) (Wolf et al., 2018). For each cell, a CMA activation score was calculated. To do so, each element of the CMA network was attributed a weight. As LAMP-2A is the rate limiting component of CMA, it was given a weight of 2. Every other element received a weight of 1. Then, every element was attributed direction score that is +1 or −1 based on the known effect of a given element on CMA activity. The score was then calculated as the weighted/directed average of expression counts of every element of the CMA network. To ease understanding of transcriptional CMA activity changes between conditions, the CMA score was expressed as fold of healthy individuals within a given cell type.
All data presented are mean±s.e.m. with *: p<0.05, **: p<0.005, **: p<0.0005. Prior to statistical testing, normality was assessed using the Shapiro Wilk test. Parameters with two groups were compared using unpaired, two-tailed t-test or Mann Whitney U test. Parameters with more than two groups were compared using one-way ANOVA and Tukey's post-hoc analysis, Kruskal-Wallis test followed by Dunn's post-hoc analysis or two-way ANOVA followed by Sidak post-hoc analysis. The number of animals used per experiment was calculated through power analysis based in previous results. Statistical analyses were performed either in GraphPad Prism 8.0 or using Python (Python software foundation v.3.7.4 available at https://www.python.org/) and the scientific python stack: scipy (v.1.3.1), numpy (v.1.17.2) (van der Walt et al., 2011), and matplotlib (v.3.1.1).
All chemical reagents and solvents were obtained from commercial sources (Aldrich, Acros, Fisher) and used without further purification unless otherwise noted. Chromatography was performed on a Teledyne ISCO CombiFlash Rf 200i using disposable silica cartridges (4, 12, and 24 g). Analytical thin layer chromatography (TLC) was performed on aluminum-backed Silicycle silica gel plates (250 μm film thickness, indicator F254). Compounds were visualized using a dual wavelength (254 and 365 nm) UV lamp, and/or staining with CAM (cerium ammonium molybdate) or KMnO4 stains. NMR spectra were recorded on Bruker DRX 300 and DRX 600 spectrometers. 1H and 13C chemical shifts (d) are reported relative to tetramethyl silane (TMS, 0.00/0.00 ppm) as internal standard or to residual solvent (CDCl3: 7.26/77.16 ppm; dmso-d6: 2.50/39.52 ppm). Mass spectra (ESI-MS) were recorded on a Shimadzu LCMS 2010EV (direct injection unless otherwise noted). High resolution mass spectra (HRMS) were recorded on an Orbitrap Velos high resolution mass spectrometer at the Proteomics Facility of Albert Einstein College of Medicine.
The following compounds, which have been previously disclosed, are used in the examples.
We generated a neuronal-specific CMA-deficient mouse model (CKL2A−/− mice) by removing L2A in excitatory neurons (expressing calcium/calmodulin-dependent protein kinase II (α-CaMKIIα)). We breed L2Aflox/flox mice with a CaMKIIα-driven Cre recombinase transgenic mouse line for selective deletion of the Lamp2 gene exon encoding for the cytosolic and transmembrane domain of the L2A protein. (
Behavioral testing revealed higher scores and faster hindlimb clasping progression, a phenotype of neurological disorder models, in L2A−/− and CKL2A−/− mice than control (CTR) littermates as early as 3 months of age (FIGS. B,C). Both mouse models showed higher latency in the negative geotaxis test, indicative of vestibular/sensorimotor dysfunction (
By 6 months of age, both mice with systemic or neuronal-specific CMA loss showed thicknesses of CA1, dentate gyrus and cortex as well as hippocampal surface undistinguishable from their respective CTR littermates (
Analysis of overall cellular proteostasis revealed lipofuscin deposits (cross-linked oxidized proteins and lipids) (
To analyze the extent of proteome compromise upon neuronal CMA blockage, we isolated the sarkosyl-insoluble fraction from the cortex of CTR and CKL2A−/− mice and performed comparative quantitative proteomics. We found a significant enrichment of proteins in the insoluble fraction of CKL2A−/− mice brains. Most insoluble proteins (76%) contained KFERQ-like motifs, mainly those that do not require posttranslational modifications for hsc70 recognition (˜52% in CKL2A−/− sarkosyl-insoluble fraction vs. ˜47% in the whole murine proteome (Kirchner et al., 2019)). Prone-to-aggregate proteins bearing KFERQ-like motifs such as α-syn, tau, UCHL1 and PARK7, displayed a shift towards insolubility in CKL2A−/− brains, whereas this was not the case for SOD1 that lacks the motif (
Taken together, these results demonstrate a profound collapse of the proteome upon neuronal CMA blockage.
Neuronal protein aggregates have been described upon blockage of macroautophagy. However, the loss of proteostasis in CMA-deficient brains was not due to macroautophagy malfunction. Contrary to the macroautophagy failure observed when all three spliced variants of LAMP2 were eliminated, steady-state levels of autophagosome components (LC3 and GATE16), macroautophagy adaptors (p62) and lysosomal membrane proteins (LAMP1) were comparable in cortex and hippocampus of L2A−/−, CKL2A−/− and their corresponding CTR littermates (
We used comparative proteomics of the insoluble proteome of CKL2A−/− and CKATG7−/− mice to elucidate the specific contribution of CMA and macroautophagy to neuronal proteostasis (
Gene-set enrichment and Enrichment Map analyses of proteins in the insoluble fractions of only CKL2A−/− or CKATG7−/− mice brains (
We experimentally confirmed that the presence of these proteins in the insoluble fraction had functional consequences in the CMA-defective neurons. Measurement of the extracellular acidification rate (ECAR) revealed pronounced decrease of glycolysis in CKL2A−/− neurons (
To further confirm the functional consequences of the shift toward insolubility of the CMA-deficient neuronal proteome, we analyzed a second protein cluster—“protein targeting and localization”—enriched in proteins related to endocytosis and to actin remodeling (
These results demonstrate that defective neuronal function upon CMA blockage is a combination of a direct impact on the solubility of the neuronal proteome and indirect effects due to the functional disruption of aggregate-entrapped CMA substrate proteins.
The alterations in proteostasis and neuronal function upon direct neuronal CMA blockage, indicate that reduced CMA activity increases vulnerability and accelerates disease progression in neurodegenerative disorders.
To test this possibility, we measured CMA in a mouse model of tau-mediated proteotoxicity expressing mutant human tau (hTauP301L) under the Thy 1.2 promotor (line pR5). We crossed hTauP301L mice with a transgenic mouse model that expresses systemically a KFERQ-tagged fluorescent protein Dendra2 (KFERQ-Dendra), recently developed by our group. This reporter protein, when targeted for degradation via CMA, highlights lysosomes as fluorescent puncta, that can be counted as read out of CMA activity. Hippocampal neurons of KFERQ-Dendra-hTauP301L mice displayed a significant reduction in the number of fluorescent puncta compared to control KFERQ-Dendra mice (
Insights on the CMA status of the neurodegenerating human brain have been so far limited to analysis of L2A expression in PD blood cells or whole brain extracts. We took advantage of a recently published single-nucleus (sn) RNAseq dataset from prefrontal cortex of 24 healthy individuals and 24 AD patients grouped according to their Braak score in 3 categories: low (Braak 0, I, II), medium (Braak III, IV) and high (Braak V, VI) (Mathys et al., Nature (2019) 570: 332-337, and extracted the expression level of every element in the CMA network (
To infer the impact of the transcriptional differences on CMA output and compare CMA activity among cell types and pathological stages, we next defined a CMA activation score. This score is a weighted average of the expression level of every element of the CMA network. Higher scores could result from (i) increased expression of effectors or positive modulators or (ii) decreased expression of negative modulators, whereas changes in opposite direction will render lower CMA activation scores. We experimentally validated this score in cultured cells exposed to pro-oxidant conditions (which activate CMA) or to a chemical CMA activator.
Using the CMA activation score in the snRNAseq dataset from the control and AD human brains, we found an inhibition of the CMA network already at early pathology stages in both excitatory and inhibitory neurons, followed by deeper inhibition at later disease stages (
The CMA activation score of excitatory neurons revealed significant negative correlation with different quantitative pathology markers such as the Braak stage (
Altogether, these findings reveal a previously unknown neuron-specific transcriptional downregulation of CMA early in AD, that could add to the direct toxic effect of pathogenic AD proteins on CMA, as reported before (Caballero et al., 2018).
To evaluate the contribution to disease progression of the observed early inhibition of CMA in AD, we imposed CMA loss to a mouse model of AD-related proteotoxicity by breeding triple transgenic (Tg) AD mice (expressing APPSwe, PS2N141I and hTauP301L) with L2A−/− mice (hereafter named Tg-L2A−/−) (
Total tau levels displayed similar progression in Tg and Tg-L2A−/− mice brains at 5, 8 and 12 months of age, while pS202/T205 tau levels were constantly higher in Tg-L2A−/− mice and both, pS422 and aggregated tau showed higher levels and faster accumulation rates (2.5 to 3.1 folds) (
To identify how CMA deficiency aggravates AD, we used comparative quantitative proteomic of brain cortex from CTR, Tg, L2A−/−, and Tg-L2A−/− mice. We found 152 differentially expressed proteins between CTR and L2A−/− mice (
Imposing CMA blockage in the context of AD-related brain proteotoxicity resulted in quantitative and qualitative proteome changes not recapitulated by any of the genotypes separately. Direct comparison of Tg-L2A−/− and L2A−/− proteomes and Tg-L2A−/− and Tg proteomes revealed little overlap in differentially expressed proteins. Gene ontology and network analysis to identify biological processes altered only in Tg-L2A−/− mice (
We reasoned that our mouse model of AD with defective CMA, may recapitulate, at least in part, human disease progression, since CMA activity will further decrease in AD patients as they age. Comparison of the proteomic changes in brains of low and high pathology controls, asymptomatic and symptomatic AD patients from the Consensus study (Johnson et al., Nature Medicine (2020) 26: 769-780) with those in our Tg-L2A−/− mice, supports this possibility as we found: (i) the highest positive correlation between proteome changes in AD patients and Tg-L2A−/− mice brains, (ii) stronger overlap between AD patients and Tg-L2A−/− mice signatures from proteome-wide transition mapping than with Tg or L2A−/− mice (
Reduced CMA may also accelerate the underlying autophagy/lysosomal found in AD brains. Contrary to Tg and L2A−/− mice, Tg-L2A−/− mice showed accumulation of p62 and GATE-16 (suggestive of reduced autophagic flux), marked increase in lysosomal hexosaminidase activity and accumulation and mislocalization of cathepsin D, as occurs in the AD brain (note that cathepsin D levels increase in homogenate but not in lysosomes). As expected, elimination of L2A alone or in the Tg background did not have major effect in levels of other CMA components.
Overall, these results support the aggravating effect of CMA loss with age on neurodegenerative disease progression due to synergistic, and not merely additive, alterations of the brain proteome.
To test if CMA activation could be protective against AD-related neuroproteotoxicity, we performed extensive medicinal chemistry on AR7, one of the generated CMA activators, to make derivatives suitable for in vivo administration (see Methods section). The derivative used in this study (CA77.1, thereafter CA) activates CMA in vitro in dose- and time-dependent manner without affecting macroautophagy (
We first administered CA to PS19 mice, which express tau with the frontotemporal dementia mutation P301S. We used a clinically relevant administration design (
Next, we used the triple transgenic mouse model of AD (Tg) (Grueninger et al., Neurobiol. Dis. (2010) 37: 294-306) to explore the effect of pharmacological activation of CMA on combined tau and Aβ proteotoxicity. Tg mice were given daily oral doses of CA (30 mg/kg body weight) for 4 months starting at 8 months of age (after symptoms' onset and when b-amyloid plaques are already detectable. CA-treated Tg mice had better visual memory, decreased anxiety- and depression-like behaviors, slower clasping progression and increased performance in horizontal grid test than those receiving the vehicle (
These results indicate that pharmacological CMA activation using a clinically relevant design has a beneficial effect on AD-related pathology.
To experimentally test the proposed protective effect of CMA activation—observed early in the disease in mice and in human plaques—and to evaluate the possible therapeutic value of CMA modulation in atherosclerosis, we directly upregulated CMA activity in mice exposed to a proatherosclerotic challenge. To that end, we used an inducible transgenic mouse model (hL2AOE), expressing the human form of LAMP-2A, which we induced after the observed drop in LAMP-2A levels in early plaques (
Our in vitro and in vivo findings support that CMA upregulation may be part of the vascular response to pro-atherosclerotic challenges. To test whether that was also the case in human atherosclerosis, we first confirmed the presence of the CMA receptor in plaque VSMC and macrophages using costaining for LAMP-2A and αSMA/CD68. Analysis of levels of LAMP-2A in human atherosclerotic plaques from asymptomatic patients at different plaque stages revealed that LAMP-2A levels at the plaque increase gradually with disease progression (graded as plaques with moderate intimal thickening (IT), pathological intimal thickening (PIT), thick fibrous cap atheroma (TkFCA) and plaques with intraplaque hemorrhage (IPH)). The increase in LAMP-2A protein levels originates mainly from LAMP-2A mRNA upregulation. In fact, LAMP-2A mRNA levels directly correlated with the size of the plaque but not the necrotic core. To determine the cell type mainly contributing to the elevated levels of LAMP-2A at the plaque, we analyzed the correlation between LAMP-2A levels and different cell types and found a direct correlation between LAMP-2A and CD68, a marker of macrophages and foam cells, in human atherosclerotic plaques. We interpreted these changes in LAMP-2A levels as an attempt of the plaque cells, mostly macrophages, to upregulate this autophagic pathway in response to the pro-atherosclerotic changes prior to clinical events, as we observed in the experimental mouse model. In fact, holistic analysis of the CMA transcriptional network using single cell RNA seq (scRNAseq) from human atherosclerotic plaques (from (8)) confirmed macrophages as the cells with the highest expression of CMA effectors (LAMP-2A and (HSC70) when compared to endothelial and smooth muscle cells from the same plaques. To evaluate possible changes of CMA after the clinical event, we performed immunoblot for LAMP2 in carotid segments from patients who suffered one or two clinical vascular events.
This revealed a significant decrease in LAMP2 levels in carotid segments from all patients who develop a second event (
Our findings suggest that reduced levels of LAMP-2A, and lower CMA activity could be a predictor of the risk of suffering a second clinical event.
We used the recently developed transgenic mouse model expressing a fluorescence reporter for CMA (KFERQ-PS-Dendra2 mice) that allows measuring CMA activity in vivo to determine the status of CMA in the vasculature and its possible changes during atherosclerotic plaque development. When KFERQ-PS-Dendra CMA substrate is delivered to lysosomes, CMA activity is detected as fluorescent puncta against the diffuse fluorescent cytosolic pattern. Using aortas from healthy mice and two-photon microscopy in fixed tissue or intravital two photon microscopy, we found fluorescent puncta in cells in the media (VSMC) and to less extent in the intima (endothelial cells). Injection of fluorescent dextran that highlights the lysosomal compartment upon internalization from the bloodstream by endocytosis demonstrated colocalization with the Dendra signal in a fraction of lysosomes, in support of active CMA in the vasculature under basal conditions. When we promoted atherosclerosis development in KFERQ-PS-Dendra2 mice through hypercholesterolemia (using injection of adeno-associated virus 8-mediated overexpression of proprotein convertase subtilisin/kexin type 9 (AAV8-PCSK9) and a high cholesterol-containing diet (Western type diet, WD) for 12 weeks), aortas from these mice revealed a marked reduction in the number of fluorescent puncta that was almost absent in the plaque (
To determine if the initial upregulation of LAMP-2A in response to the dietary challenge was protective and whether reduced CMA contributes to disease progression, we used a mouse model with systemic blockage of CMA (constitutive knock-out for LAMP-2A, L2AKO. At 3 months of age, L2AKO mice on chow diet display slightly lower body weight and circulating total cholesterol levels than wild-type (WT) littermates. When L2AKO mice were fed WD for 12 weeks, we observed a marked increase in total circulating cholesterol and triglyceride (TG) levels (
Overall, reduced CMA activity associates with many aspects of more severe atherosclerotic pathology supporting an anti-atherosclerotic protective function for CMA.
To determine the basis for the protective effect of CMA against atherosclerosis, and because of the previously described regulation of hepatic glucose and lipid metabolism by CMA, we evaluated metabolic parameters shown to be major risks factors for CVD. We found that L2AKO mice gained 50% more body weight than the WT group during the 12 weeks of WD, mostly due to a higher fat mass content. Indirect calorimetry revealed that the increased adiposity of L2AKO mice did not originate from higher food consumption, but it could be explained by reduced energy expenditure and less physical activity. The decrease in respiratory exchange ratio (RER)—6 indicative of lipid use as energy—observed in WT mice on WD was significantly more pronounced in L2AKO mice, suggesting impaired carbohydrate utilization in these mice. Indeed, L2AKO mice showed marked hyperinsulinemia and increased insulin resistance, typical risk factors for CVD. Circulating levels of the prothrombotic and pro-fibrotic cytokine plasminogen activator inhibitor type 1 (PAI-1) were also significantly higher in L2AKO mice. These findings support that loss of CMA accentuates the systemic derangements in metabolism and coagulation imposed by the WD, thus rendering organisms more prone to atherosclerosis. CMA blockage promotes VSMC dedifferentiation. Whereas circulating cholesterol levels in WT mice show the previously described correlation with different plaque properties, such correlations are lost in L2AKO mice. This suggests that factors other than systemic metabolic changes also contribute to the higher vulnerability of L2AKO mice to atherosclerosis. This motivated us to investigate whether local changes of CMA in the vasculature could contribute to disease progression.
We first examined CMA in primary cultured VSMC exposed to a physiological lipid challenge (LDL loading) and found a dose-dependent upregulation of CMA followed by a gradual decrease once toxic concentrations of LDL are reached. Exposure of L2AKO VSMC to fluorescent LDL (diLDL) resulted in higher intracellular lipid accumulation (
Loading with LDL induced changes in genes related to lipid metabolism in both genotypes, but we identified quantitative differences in this response. Thus, using Ingenuity Pathway Analysis (IPA), we found that L2AKO cells have a defective response to the lipid challenge with reduced upregulation of genes involved in the cholesterol pathway and display cholesterol as one of the top molecules upregulated in these cells. The immune component of the response of VSMC to lipids is also different in L2AKO cells. While WT cells orchestrate a well characterized inflammatory response, the immune response of L2AKO cells is mainly composed of genes related to leucocyte activation and cell migration. Differential gene expression analysis and gene set enrichment upon lipid loading also identified gene nodes unique for L2AKO cells related with cell death and cellular response to stress, including the response to DNA damage, which we experimentally confirmed to be significantly increased in these cells. These findings support that failure to activate CMA in VSMC makes them unable to adapt to the environmental lipid challenge, as previously described also in CMA-deficient hepatocytes.
Analysis of upstream regulators of the group of genes differentially expressed in L2AKO VSMC revealed as the top change a significant (p<3.13×10−46) downregulation of the tumor protein 53 (p53) signaling pathway. Immunoblot against different components of the p53 signaling pathways confirmed markedly reduced levels of p53 protein and of the cyclin-dependent kinase inhibitor 1A (p21) in L2AKO VSMC, whereas cyclin-dependent kinase inhibitor 1B (p27) content was higher in these cells compared with WT. Considering the well-characterized role of p53 as anti-apoptotic molecule in response to lipid challenges, the identified defect in p53 signaling in L2AKO VSMC provides an explanation for their higher death count (
The presence of macrophages in the plaque and their associated inflammatory phenotype influence plaque fate. Therefore, we next set to investigate the consequences of CMA blockage in macrophage function using in vitro protocols for polarization of bone marrow-derived macrophages (BMDM) to mimic the plaque pro-inflammatory phenotype of these cells (IFNγ+LPS). We found that CMA-defective BMDM, when stimulated with IFNγ+LPS, show a stronger pro-inflammatory profile (higher inducible nitric oxide synthase, iNOS, and cytochrome c oxidase 2 (COX2) levels (
We aimed to identify the subset of the proteome that, by not undergoing degradation through CMA, could be behind this exacerbated inflammatory phenotype seen in the L2AKO BMDM. Thus, we isolated the pool of lysosomes usually active for CMA, those that contain high levels of luminal HSC70, from WT and L2AKO BMDM, untreated (CTRL) or stimulated with IFNγ+LPS. In half of the cultures, we inhibited lysosomal proteolysis to discriminate proteins undergoing degradation inside lysosomes from lysosomal resident proteins and subjected the samples to comparative quantitative proteomics (21). About 45% of the proteins were constitutive lysosomal components, not degraded in lysosomes in resting or stimulated BMDM. CMA substrates are defined as those proteins undergoing degradation in lysosomes in a LAMP-2A-dependent manner. Stimulation with IFNγ+LPS resulted in an increase of lysosomal protein degradation, mostly of CMA substrates (46% increase in CMA substrates vs. only 15% increase in non-CMA lysosomal substrates;
Overall, our findings support that CMA contributes to the remodeling of the proteome induced by macrophage stimulation, and that defective CMA in these cells promotes a more proinflammatory phenotype.
This application claims priority of U.S. Provisional Appl. No. 63/177,504 filed Apr. 21, 2021, and U.S. Provisional Appl. No. 63/323,942 filed Apr. Mar. 25, 2022.
Filing Document | Filing Date | Country | Kind |
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PCT/US2022/025757 | 4/21/2022 | WO |
Number | Date | Country | |
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63177504 | Apr 2021 | US | |
63323942 | Mar 2022 | US |